Polymer engineered regenerating bioscavengers

ABSTRACT

Embodiments of the invention provide at least one polymer covalently conjugated to an esterase. The at least one polymer includes a plurality of oxime functional groups.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/026,093, filed Mar.30, 2016, which is a National Stage application under 35 U.S.C. § 371 ofInternational Application No. PCT/US14/59171 having an InternationalFiling Date of Oct. 3, 2014, which claims the benefit of priority fromU.S. Provisional Application Ser. No. 61/961,097, filed Oct. 3, 2013.The disclosures of the prior applications are considered part of (andare incorporated by reference in) the disclosure of this application.

FIELD OF THE INVENTION

This invention describes the development of a broad spectrumbiocatalytic scavenger for use as an organophosphate (OP) detoxifyingagent.

BACKGROUND OF THE INVENTION

The dramatic tragedy in the reemergence of chemical weapons use in Syriahighlights like never before the urgent need to provide effectivemedical countermeasures for nerve agent poisoning. Less talked of, buteven more urgent, is the need for countermeasures againstorganophosphate (OP) pesticide poisoning that kills hundreds ofthousands world-wide every year. Although most OP pesticides were bannedfor use in the US, they still exist and are produced in Kilo-tons inother countries and are easily accessible. Some of them are highly toxicas witnessed by the fatal poisoning of school children in India in July2013 caused by monocrotophos contamination of cooking oil. OP pesticidesare a major worldwide health problem, even without accidental ordeliberate release with OP self-poisoning (suicide), responsible for200,000 deaths a year worldwide. Eddleston M, Buckley N A, Eyer P,Dawson A H. Management of acute organophosphorus pesticide poisoning.Lancet, 371: 597-607 (2008). Further, these poisonings are difficult totreat with the current standards of therapy.

SUMMARY OF THE INVENTION

The present investigation represents a solution for the need forcountermeasures against organophosphate pesticide poisoning. A broadspectrum regenerating scavenger for use as an OP compound detoxifyingagent following acute exposure is described herein.

Accordingly, the present disclosure may include various aspects of acomposition and methods for making and using the composition. Forexample, in various aspects, the invention described herein provides acomposition comprising at least one polymer covalently conjugated to anesterase. The at least one polymer may comprise a plurality of oximefunctional groups.

In various aspects, the invention may include a composition wherein theesterase may comprise a member selected from the group consisting ofacetylcholinesterase and butyrylcholinesterase. In various aspects, theinvention may include a composition wherein the esterase may notcomprise acetylcholinesterase. In various aspects, the invention mayinclude a composition as provided in any of the aspects describedherein, wherein the esterase may not include butyrylcholinesterase.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theplurality of oxime functional groups may comprise alkyne derivatives of2-pyridine aldoxime.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theplurality of oxime functional groups may comprise an aldoxime. Invarious aspects, the invention may include a composition as provided inany of the aspects of the composition described herein, wherein theplurality of oxime functional groups may comprise a ketoxime. In variousaspects, the invention may include a composition as provided in any ofthe aspects of the composition described herein, wherein the pluralityof oxime functional groups may comprise at least one bis-pyridiniumoxime.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theat least one polymer covalently conjugated to the esterase may comprisea long lived covalent conjugate.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein thelong lived covalent conjugate may comprise a conjugate that ismaintained in the body for a time period ranging from at least one 24hour day and preferably for more than about one day to more than aboutone week. The long lived covalent conjugate may be maintained in thebody for a period of time of at least one week, or at least two weeks,and preferably for a period of time sufficient to eliminate orsignificantly reduce the inhibiting function of the OP toxin.

In various aspects, of the invention, any of the aspects of thecomposition described herein may comprise at least one polymer which mayhave at least one environmentally responsive monomer.

In various aspects of the invention, any of the aspects of thecomposition described herein may comprise, at least one polymer having apolymer length of two repeat units, and preferably greater than tworepeat units, and more preferably a polymer length ranging from aminimum of at least 2 monomer repeats to about 1000 monomer repeats. Incertain aspects of the composition, there may be greater than 1000monomer repeats. The only upper limit on the number or length ofmonomers is that number or length that will avoid hindering contact ofthe oxime functional group or groups sufficient to interact with theactive site of the enzyme for neutralization of the inhibiting moiety.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theat least one polymer may be a co-polymer that may comprise at least twodifferent monomers, wherein at least one monomer may comprise a memberselected from the group consisting of aldoximes, ketoximes,muco-adhesion monomers, polyethylene glycol, bis-pyridinium oximes,N,N-dimethylacrylamide, N-isopropylacrylamide, (meth)acrylate,N,N-dimethylaminoethyl methacrylate, carboxyl acrylamide,2-hydroxylethylmethacrylate, N-(2-hydroxypropyl)methacrylamide,quaternary ammonium monomers, sulfobetain methacrylate, oligo(ethyleneglycol) methyl ether methacrylate, 2-PAM monomers, 4-PAM monomers,Clickable azide monomers, and combinations thereof.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein thereare a plurality of polymers each covalently conjugated to the esterase,and each polymer may comprise a plurality of monomer units wherein atleast one said monomer unit comprises an oxime functional group.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein aplurality of monomer units of each polymer comprises an oxime functionalgroup.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theplurality of polymers comprises co-polymers, each co-polymer comprisingat least two different monomers. At least one such monomer comprises amember selected from the group consisting of aldoximes, ketoximes,muco-adhesion monomers, polyethylene glycol, bis-pyridinium oximes,N,N-dimethylacrylamide, N-isopropylacrylamide, (meth)acrylate,N,N-dimethylaminoethyl methacrylate, carboxyl acrylamide,2-hydroxylethylmethacrylate, N-(2-hydroxypropyl)methacrylamide,quaternary ammonium monomers, sulfobetain methacrylate, oligo(ethyleneglycol) methyl ether methacrylate, 2-PAM monomers, 4-PAM monomers,Clickable azide monomers, and combinations thereof.

In various aspects, the invention may include a composition as providedin any of the aspects of the composition described herein, wherein theplurality of polymers comprises a plurality of co-polymers and aplurality of homopolymers. Each co-polymer of the plurality ofco-polymers may comprise at least two different monomers, wherein atleast one monomer comprises a member selected from the group consistingof aldoximes, ketoximes, muco-adhesion monomers, polyethylene glycol,bis-pyridinium oximes, N,N-dimethylacrylamide, N-isopropylacrylamide,(meth)acrylate, N,N-dimethylaminoethyl methacrylate, carboxylacrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and combinations thereof. Each homopolymer of the plurality ofhomopolymers may comprise a member selected from the group consisting ofaldoximes, ketoximes, muco-adhesion monomers, polyethylene glycol,bis-pyridinium oximes, N,N-dimethylacrylamide, N-isopropylacrylamide,(meth)acrylate, N,N-dimethylaminoethyl methacrylate, carboxylacrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and combinations thereof.

In certain aspects, the invention may include a composition comprising abioconjugate which comprises an esterase and at least one polymercovalently conjugated to the esterase. In various aspects, of thebioconjugate, the at least one polymer comprises a plurality of oximefunctional groups; and at least one oxime functional group of theplurality of oxime functional groups is positioned to react, in use,with a phosphoryl functional group when an inhibitor having a phosphorylfunctional group attaches to an active site of the esterase.

In certain aspects, the invention may include a composition according toany of the aspects of the composition described herein wherein the atleast one oxime functional group is positioned to exert a nucleophilicattack on a phosphoryl functional group when, in use, an inhibitorhaving a phosphoryl functional group attaches to an active site of theesterase, said nucleophilic attack resulting in the removal of thephosphoryl functional group from the active site.

In certain aspects, the invention may include a composition according toany of the aspects of the composition or bioconjugate described herein,wherein the at least one polymer of the bioconjugate may comprise aflexibility sufficient to react, in use, with a phosphoryl functionalgroup when an inhibitor having a phosphoryl functional group attaches tothe active site of the esterase.

In certain aspects, the bioconjugate may include any of the aspects ofthe composition described herein, such as, for example, wherein theesterase is a member selected from the group consisting ofacetylcholinesterase, butyrylcholinesterase, and chymotrypsin.

In certain aspects, the invention may include a bioconjugate comprisingany of the aspects of the composition described herein, such as, forexample, the at least one oxime functional group, wherein suchfunctional group comprises one or more of alkyne derivatives of2-pyridine aldoxime, an aldoxime or a ketoxime, or at least onebis-pyridinium oxime. In certain aspects, the invention may include abioconjugate comprising any of the aspects of the composition describedherein, such as, for example, wherein the at least one polymer comprisesat least one environmentally responsive monomer, and the at least onepolymer may comprise a co-polymer comprising at least two differentmonomers, wherein at least one monomer comprises a member selected fromthe group consisting of aldoximes, ketoximes, muco-adhesion monomers,polyethylene glycol, bis-pyridinium oximes, N,N-dimethylacrylamide,N-isopropylacrylamide, (meth)acrylate, N,N-dimethylaminoethylmethacrylate, carboxyl acrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and combinations thereof.

In various aspects, the composition according to any of the aspectsdescribed herein may function to reduce or eliminate the effectivenessof an inhibitor which may comprise a member selected from the groupconsisting of VX, Sarin, Soman, DFP, Paraoxon, Parathion, V classes ofOP nerve agents, G classes of OP nerve agents, and combinations thereof.

In another aspect, the invention described herein may include a methodcomprising administering a bioscavenger according to any of the aspectsor combination of aspects of the composition or the bioscavengerdescribed herein to an individual suffering from organophosphate toxinexposure. The individual is preferably a mammal and may be a humanvictim of OP exposure. The bioscavenger used in the method may comprisefor example, at least one polymer covalently conjugated to an esterase,the at least one polymer comprising a plurality of oxime functionalgroups. The method may further comprise following administration andupon exposure of the bioscavenger to the organophosphate toxin, reactingat least one of the plurality of oxime functional groups with at leastone covalently inhibited residue of the esterase to detoxify theorganophosphate toxin and regenerate the bioscavenger.

In various aspects, upon exposure of the bioscavenger to theorganophosphate toxin, the method as described herein may furthercomprise reacting at least one of the plurality of oxime functionalgroups with at least one covalently inhibited residue of the esterase todetoxify the organophosphate toxin and regenerate the bioscavenger.

In certain aspects, the plurality of oxime functional groups of thebioscavenger used in any aspect of the method may comprise an oximefunctional group positioned to exert a nucleophilic attack on aphosphoryl functional group when an inhibitor having a phosphorylfunctional group attaches to an active site of the esterase. Thenucleophilic attack results in the removal of the phosphoryl functionalgroup from the active site.

In various aspects, the invention described herein may be in the form ofa bioscavenger comprising any aspect or combination of aspects of thecomposition described herein. For example, the bioscavenger may compriseat least one polymer covalently conjugated to an esterase, the at leastone polymer comprising a plurality of oxime functional groups, whereinat least one of the plurality of oxime functional groups is positionedto exert a nucleophilic attack on a phosphoryl functional group when, inuse, a phosphoryl functional group is covalently attached to an activesite of the esterase effecting removal of the phosphoryl functionalgroup from the active site and regeneration of the bioscavenger. The atleast one polymer of the bioconjugate may comprise any one or more ofthe aspects of the polymers described herein, and may comprise any oneor more of the aspects of the oxime functional groups described herein,and as described above the esterase of the bioconjugate may compriseacetylcholinesterase, butyrylcholinesterase, or chymotrypsin. In certainaspects of the bioscavenger, one or two of acetylcholinesterase,butyrylcholinesterase, or chymotrypsin may be absent from thecomposition of the bioscavenger. The oxime functional group of thebioscavenger is preferably positioned to exert a nucleophilic attack ona phosphoryl functional group when, in use, an inhibitor having aphosphoryl functional group attaches to an active site of the esterase,said nucleophilic attack resulting in the removal of the phosphorylfunctional group from the active site, preferably resulting in theremoval of the phosphoryl functional group from the active site.

In one aspect, the invention described herein provides a method forbiocatalytic scavenging by using polymer-based protein engineering toconjugate oxime containing polymers directly to enzymes providing atethered pseudo-cofactor. As used herein, a “pseudo-factor” is asynthetic compound that assists an enzyme in completing a catalyticcycle and which may or may not be consumed in the process. Judiciousdesign of the polymer conjugates and the exquisite synthetic controlafforded by atom transfer radical polymerization (ATRP) can provideengineered proteins of broad variation. Averick S E, Konkolewicz D,Matyjaszewski K., Aqueous ARGET ATRP, Macromolecules, 45(16):6371-9(2012); Simakova A, Park S, Konkolewicz D, Magenau A I D, Mehl R A, etal., ATRP under Biologically Relevant Conditions: Grafting from aProtein, ACS Macro Letters, 1(1):6-10. (2012); Konkolewicz D, Magenau AJ D, Averick S E, Simakova A, He H, Matyjaszewski K., ICAR ATRP with ppmCu Catalyst in Water, Macromolecules, 45(11):4461-8 (2012); Magenau A JD, Averick S E, Simakova A, He H, Matyjaszewski K. ICAR ATRP with ppm CuCatalyst in Water, Macromolecules, 45(11):4461-8 (2012); MatyjaszewskiK, Atom Transfer Radical Polymerization: From Mechanisms toApplications, Isr J Chem.; 52(3-4):206-20 (2012); Matyjaszewski K, AtomTransfer Radical Polymerization (ATRP): Current Status and FuturePerspectives, Macromolecules, 45(10):4015-39 (2012).

One of the unmet needs in the treatment of organophosphate (OP)intoxication is a broad spectrum non-stoichiometric antidotal scavengeruseful in single occurrence or mass casualty scenarios. Toward that end,the present invention addresses these issues and describes thedevelopment of a broad spectrum regenerating scavenger for use as an OPcompound detoxifying agent following acute exposure.

The role of acetylcholinesterase (AChE) in nerve and muscle is toterminate the neurotransmission signal exerted by acetylcholine by rapidhydrolysis at a sub-millisecond time scale. The irreversible inhibitionof AChE by OP nerve agents and pesticides leads to persistent severetoxic symptoms and death caused by excess acetylcholine in cholinergicsynapses in the peripheral and central nervous system. The ability ofmany OP compounds (e.g., VX, Sarin, Soman, DFP, Paraoxon, G-agents,V-agents, Parathion, and the like) to irreversibly inhibit AChEcatalytic activity is due to the hydrolysis of the OP compound withinthe active site of the enzyme with the concomitant formation of acovalent bond between the phosphoryl moiety of the OP and the catalyticserine residue within the enzyme active site. The inhibition of thevarious members of the cholinesterase family by OP compounds isdependent upon how the three dimensional structure of a given OP fitswithin the three dimensional structure of the enzyme active site. All ofthe cholinesterases are venerable to inhibition by one or more OPcompound. Covalent modification by organophosphate inhibition preventsAChE from completing a catalytic cycle thus locking it in anintermediate catalytically inactive state. Enzymes within the serineprotease family (e.g., chymotrypsin and trypsin) have active sitearchitectures that are similar to the cholinesterases and similarlyinhibited by some organophosphate compounds. Green A L and Nicholls J.,The Reactivation of Phosphorylated Chymotrypsin, Biochem J., 72(1):70-75 (1959). Some molecules with oxime functionality, For example,2-pyridine aldoxime (2-PAM), toxogonin (obidoxime), MMB-4 (methoxime)and HI-6 (asoxime chloride), constitute the very few clinically approvedantidotes to OP intoxication. The antidotal oximes function by reactingwith the phosphonylated serine in the enzyme active site regeneratingenzyme activity. For the purposes of discussion consider an oximereaction with an OP-inhibited enzyme to be the final step in thecatalytic hydrolysis of OP toxins (pesticides and nerve agents) in thatit releases a non-toxic alkyl-phosphonate as a product of the reactionand returns the enzyme to its active state.

Current antidote therapies couple oxime drugs with atropine (amuscarinic cholinergic antagonist) and anticonvulsants. While thequaternary oximes are generally considered effective in the short term,the rapid clearance of oximes such as 2-PAM from circulation in the bodycombined with the long residence times and relatively slow adsorptionand distribution rates of OP nerve agents and pesticides especiallyfollowing dermal (OP pesticides and VX) or buccal exposure (pesticides)reduces their effectiveness in cases of single occurrence or masscontamination and intoxication events where individualized continuouscare is not practical.

It should be noted that the current generation of therapeutic oximes arestoichiometric chemical scavengers as they are consumed in theirreaction with the OP. The kinetics of oxime-induced reactivation andrapid AChE inhibition by OP compounds are not compatible with longintervals between oxime dosing. While oxime onset time is relativelyfast, the retention time in blood is short after a single intra-muscularoxime injection as the elimination from blood to urine of 2-PAM has at ½of just 60 minutes. On the other hand, the time of peak ChE inhibitioncaused by nerve agents in the peripheral nervous system and the centralnervous system occurs at around 1 hour, where 50% of the oxime isalready gone. Thus, there is a need for detoxifying agents that cancover the time span of the immediate emergency treatment with continuousdetoxifying treatment without the logistic burden of applying repeatedinjections of oximes.

In order to meet the need for detoxifying agents that can cover the timespan of both immediate and continuous treatment, the compositions of thepresent invention, in at least one or more aspects, comprise esterase,such as cholinesterases (ChEs), covalently conjugated with polymerbrushes composed of tethered oxime side chains, thereby converting theminto a wide spectrum of regenerating OP-degrading enzymes. The tetheredoxime polymers act as pseudo-prosthetic groups allowing a completecatalytic cycle of the OP substrate by reactivating the OP-inhibitedenzyme.

Additionally, because oximes can also react directly with free OPcompounds in solution, the enzyme-polyoxime conjugates can increase thelifetime of the scavenger activity and act as active nucleophiles thatdirectly attack OP molecules resulting in their detoxification. Thus,the enzyme polyoxime conjugate also functions as a long lived oximedelivery system in that the enzyme can delay clearance of the oximeantitoxin from the body. For example, an esterase polymer conjugate canresult in the long lived presence of the oxime over a period rangingfrom at least about one day to about a week, and preferably more thanone week.

It should be understood that this disclosure is not limited to thevarious aspects or embodiments disclosed in this Summary, and it isintended to cover modifications that are within the spirit and scope ofthe invention, as defined by the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The characteristics and advantages of the present disclosure may bebetter understood by reference to the accompanying figures.

FIG. 1 is a schematic illustration of a conventional stoichiometricbioscavenger showing the stoichiometric binding and sequestering of aninhibitor (I) by formation of an enzyme (E)/inhibitor complex (EI).Additionally shown in FIG. 1 is the regeneration of the enzyme (E) withregenerating molecule (R) to form an inhibitor/regenerating moleculecomplex (IR).

FIG. 2 is a schematic illustration of a regenerating bioscavenger enzyme(ER4) that cleaves an organophosphate toxin substrate (I) into non-toxicmolecules (Io).

FIGS. 3A-3F are schematic diagrams and characterization data verifyingthe synthesis of an acetylcholinesterase poly-2-PAM covalent conjugate.FIG. 3A is a schematic diagram of the synthesis of a bromine containingpolymer. FIG. 3B is an ¹H NMR spectrum verifying the structure of thebromine containing polymer of FIG. 3A.

FIG. 3C is a schematic of the quaternization of the polymer of FIGS. 3Aand 3B with 2-PAM. FIG. 3D is an ¹H NMR spectrum verifying the structureof the 2-PAM containing polymer product of FIG. 3C. FIG. 3E is aschematic of the generation of an NHS group on the 2-PAM containingpolymer of FIGS. 3C and 3D. FIG. 3F is a schematic of the conjugation ofthe 2-PAM containing polymer of FIG. 3E with AChE.

FIGS. 4A and 4B are schematic diagrams and characterization dataverifying the synthesis of an acetylcholinesterase-sulfonate polymerconjugate. FIG. 4A is a schematic diagram illustratingacetylcholinesterase polysulfonate covalent conjugate formation by“grafting from” or “surface initiated” atom transfer radicalpolymerization (ATRP). FIG. 4B is an ¹H NMR spectrum verifying thestructure of the acetylcholinesterase-sulfonate polymer conjugate ofFIG. 4A.

FIG. 5 is data of an enzyme activity assay indicating the percentacetylcholinesterase activity recovered (post inhibition with paraoxon)over time showing self-reactivation of the inhibitedacetylcholinesterase poly-2-PAM covalent conjugate of FIG. 3F ascompared to the native acetylcholinesterase.

FIGS. 6A-6E are schematic diagrams and characterization data verifyingthe synthesis of an acetylcholinesterase poly-DMAA-2-PAM covalentconjugate synthesized using ATRP. FIG. 6A is a schematic of “surfaceinitiated” ATRP of DMAA and an azide monomer from an AChE-initiatorconjugate. FIG. 6B is an ¹H NMR spectrum verifying the structure of theAChE-PDMAA/Azide conjugate of FIGS. 6A and 6B. FIG. 6C is a schematic ofthe “Click” chemistry addition of an alkyne-2-PAM reagent to theAChE-PDMAA/Azide conjugate of FIG. 6C. FIG. 6D is an ¹H NMR spectrumverifying the structure of the AChE-PDMAA/2-PAM conjugate of FIG. 6C.FIG. 6E is an ¹H NMR spectrum verifying the structure of thealykyne-2-PAM reagent used in FIG. 6C in the synthesis of theAChE-PDMAA/2-PAM conjugate.

FIGS. 7A and 7B are schematic diagrams contrasting the predictedreactivation pathways for enzyme polymer attached aldoximes andketoximes.

FIG. 8 is data showing enzyme activity of AChE-polymer conjugates. Theenzyme activity was shown to be dependent on the chemistry of theattached polymer.

FIG. 9 is data showing enzyme activity of a chymotrypsin-polymerconjugate.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a regenerating bioscavenger fordetoxification against organophosphate (OP) poisoning.

Various embodiments or aspects of the invention are described andillustrated in this specification to provide an overall understanding ofthe structure, function, operation, manufacture, and use of thedisclosed compositions, systems, and methods. It is understood that thevarious embodiments or aspects described and illustrated in thisspecification are non-limiting and non-exhaustive. Thus, the inventionis not limited by the description of the various non-limiting andnon-exhaustive aspects or embodiments disclosed in this specification.Rather, the invention is defined solely by the claims. The features andcharacteristics illustrated and/or described in connection with variousaspects or embodiments may be combined with the features andcharacteristics of other aspects or embodiments. Such modifications andvariations are intended to be included within the scope of thisspecification. As such, the claims may be amended to recite any featuresor characteristics expressly or inherently described in, or otherwiseexpressly or inherently supported by, this specification. The variousaspects or embodiments disclosed and described in this specification cancomprise, consist of, or consist essentially of, or be characterized bythe features and characteristics as variously described herein.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing definitions, statements, orother disclosure material expressly set forth in this specification. Assuch, and to the extent necessary, the express disclosure as set forthin this specification supersedes any conflicting material incorporatedby reference herein. Any material, or portion thereof, that is said tobe incorporated by reference into this specification, but whichconflicts with existing definitions, statements, or other disclosurematerial set forth herein, is only incorporated to the extent that noconflict arises between that incorporated material and the existingdisclosure material. Applicant reserves the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

Reference throughout this specification to “various aspects” or “variousembodiments,” or the like, means that a particular feature orcharacteristic may be included in an aspect or embodiment. Thus, use ofthe phrase “in various aspects or embodiments,” or the like, in thisspecification does not necessarily refer to a common aspect orembodiment, and may refer to different aspects and/or embodiments.Further, the particular features or characteristics may be combined inany suitable manner in one or more aspects or embodiments. Thus, theparticular features or characteristics illustrated or described inconnection with various aspects or embodiments may be combined, in wholeor in part, with the features or characteristics of one or more otheraspects or embodiments without limitation. Such modifications andvariations are intended to be included within the scope of the presentspecification.

In this specification, other than where otherwise indicated, allnumerical parameters are to be understood as being prefaced and modifiedin all instances by the term “about”, in which the numerical parameterspossess the inherent variability characteristic of the underlyingmeasurement techniques used to determine the numerical value of theparameter. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter described in the present description should atleast be construed in light of the number of reported significant digitsand by applying ordinary rounding techniques.

Also, any numerical range recited in this specification is intended toinclude all subranges of the same numerical precision subsumed withinthe recited range. For example, a range of 1.0 to 10.0″ is intended toinclude all sub-ranges between (and including) the recited minimum valueof 1.0 and the recited maximum value of 10.0, that is, having a minimumvalue equal to or greater than 1.0 and a maximum value equal to or lessthan 10.0, such as, for example, 2.4 to 7.6. Any maximum numericallimitation recited in this specification is intended to include alllower numerical limitations subsumed therein and any minimum numericallimitation recited in this specification is intended to include allhigher numerical limitations subsumed therein. Accordingly, Applicantreserves the right to amend this specification, including the claims, toexpressly recite any sub-range subsumed within the ranges expresslyrecited herein. All such ranges are intended to be inherently describedin this specification such that amending to expressly recite any suchsub-ranges would comply with the applicable disclosure requirements.

The grammatical articles “one”, “a”, “an”, and “the”, as used in thisspecification, are intended to include “at least one” or “one or more”,unless otherwise indicated. Thus, the articles are used in thisspecification to refer to one or more than one (i.e., to “at least one”)of the grammatical objects of the article. By way of example, “acomponent” means one or more components, and thus, possibly, more thanone component is contemplated and may be employed or used in animplementation of the described embodiments. Further, the use of asingular noun includes the plural, and the use of a plural noun includesthe singular, unless the context of the usage requires otherwise.

“Bound,” “bind”, “binding”, “associated with”, or “attachment”,“attached to” and the like as used herein with respect to thecomposition, and substituents, groups, moieties, and the like of thecomposition or the OP as described herein means, unless otherwisestated, covalent or non-covalent binding, including without limitation,the attractive intermolecular forces between two or more compounds,substituents, molecules, ions or atoms that may or may not involvesharing or donating electrons. Non-covalent interactions may includeionic bonds, hydrophobic interactions, hydrogen bonds, van der Waalsforces (dispersion attractions, dipole-dipole and dipole-induced dipoleinteractions), intercalation, entropic forces, and chemical polarity.

As used herein, the term “non-toxic” refers to materials that arechemically and/or “biologically inert” or inactive with respect tobiological organisms.

As used herein the term “polymer length” refers to the length of thepolymer as a result of the number of monomers incorporated therein. A“monomer” is a molecule that may bind chemically to other molecules toform a polymer.

As used herein the term “active oxime identity” refers to the identityof an oxime functional group that is capable of interacting withmolecules within the active site of an enzyme.

As used herein the term “catalyst” refers to a substance that can causea change in the rate of a chemical reaction without itself beingconsumed in the reaction; the changing of the reaction rate by use of acatalyst is called catalysis.

As used herein the term “enzyme” refers to any of a group of catalyticproteins that are produced by living cells and that mediate and promotethe chemical processes of life without themselves being altered ordestroyed. Consonant with their role as biological catalysts, enzymesshow considerable selectivity for the molecules upon which they act(called substrates). As used herein, the terms “active site” and “enzymeactive site” refers to a specific region of an enzyme where a substratebinds and catalysis takes place (binding site).

As used herein the term “inhibitor” refers to a substance thatdiminishes the rate of a chemical reaction often by binding within theactive site in a process is referred to as “inhibition.” Inenzyme-catalyzed reactions an inhibitor frequently acts by binding tothe enzyme, in which case it may be referred to as an “enzymeinhibitor.”

As used herein, the term “bioscavenger” refers to molecules and proteinsthat function to either stoichiometrically bind and sequester theinhibiting molecule(s) or by catalytically or regeneratively cleavingthe inhibiting substrate or inhibiting molecules into non-toxicproducts. The term “stoichiometric bioscavenger(s)” refers to thosebioscavenger molecules and proteins that function to stoichiometricallybind and sequester inhibiting molecule(s). Likewise, the term“regenerating bioscavenger(s)” or “regenerative bioscavenger(s)” refersto those bioscavenger molecules and proteins that function by cleavingthe inhibiting substrate or inhibiting molecules into a non-toxicproduct.

As used herein the term “alkyl” refers to a straight-chained or branchedhydrocarbon. Examples of alkyl groups include methyl, ethyl, n-propyl,isopropyl, tert-butyl, and n-pentyl. Similarly, the term “alkenyl” or“alkynyl” refers to a straight-chained or branched hydrocarboncontaining one or more C═C double bonds or one or more C≡C triple bonds.

As used herein the term “free ChE,” “free enzyme,” “free esterase,”“free AChE,” or the like refers to native-type enzymes that are notmodified by polymers, radicals, combinations thereof, or the like. Asused herein the term “free oxime,” or “free oximes” refers to moleculesincluding an oxime functional group that are not conjugated to an enzymeor polymer and are circulating in solution.

As used herein the term “functional group” refers to specific groups ofatoms or bonds within molecules that are responsible for thecharacteristic chemical reactions of those molecules. As used herein theterm “phosphoryl functional group” refers to derivatives of phosphoricacid.

As used herein the term “oxime” refers to a chemical compound belongingto the imines, with the general formula R1R2C═NOH, where R1 is anorganic side-chain and R2 may be hydrogen, forming an aldoxime, oranother organic group, forming a ketoxime. 0-substituted oximes form aclosely related family of compounds. Amidoximes are oximes of amideswith general structure RC(═NOH)(NRR′). Oximes are usually generated bythe reaction of hydroxylamine and aldehydes or ketones. As used hereinthe term “pseudo-catalytic” refers to enzyme conjugated oximes thatgenerate a further oxime upon hydrolysis.

As used herein the term “esterase” refers to a hydrolase enzyme thatsplits esters into an acid and an alcohol in a chemical reaction withwater called hydrolysis. Cholinesterase is a family of esterasesincluding acetylcholinesterase and butyrylcholinesterase.Acetylcholinesterase (AChE) is an enzyme that degrades (through itshydrolytic activity) the neurotransmitter acetylcholine, producingcholine and an acetate group. Butyrylcholinesterase (BChE or BuChE) is anon-specific cholinesterase enzyme that hydrolyses many differentcholine esters.

As used herein an “enzyme polyoxime conjugate,” and “enzyme polymerconjugate” refers to an enzyme or esterase that has been modified toinclude a polymer or polyoxime into its protein structure. The esterasepolymer conjugate described can be in the free form or in the form ofsalt, if applicable. A salt, for example, can be formed between an anionand a positively charged group (e.g., amino) on a protein-polymerconjugate of this invention. Suitable anions include chloride, bromide,iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate,trifluoroacetate, and acetate. Likewise, a salt can also be formedbetween a cation and a negatively charged group (e.g., carboxylate) on aprotein-polymer conjugate of this invention. Suitable cations includesodium ion, potassium ion, magnesium ion, calcium ion, and an ammoniumcation such as tetramethylammonium ion. In addition, the esterasepolymer conjugate may have one or more double bonds, or one or moreasymmetric centers. Such a conjugate can occur as racemates, racemicmixtures, single enantiomers, individual diastereomers, diastereomericmixtures, and cis- or trans- or E- or Z-double bond isomeric forms.

As used herein, the term “long-lived” refers to a period of time that isgreater than about one day. As used herein the term “maintained in thebody” refers to the maintenance of the integrity of the chemicalstructure of the covalent conjugate without suffering biological orchemical degradation.

As used herein, the term “environmentally responsive” monomer refers toa monomer comprising molecules that react to a change in anenvironmental stimulus such as a change in pH, a change in light energy,a change in the electrical charge, a change in temperature, a change inpressure, or the like.

As used herein, the term “flexibility” refers to a physical ability of apolymer, monomer, co-polymer, or the like, to bend without incurringstructural damage.

As used herein, the term “bioconjugate” refers to the product of theformation of a covalent link between two biomolecules or between abiomolecule and a non-biomolecule. The term “biomolecules” or“biomolecule” refers to any molecule produced by a living organism.

One conventional investigational approach has been the use ofstoichiometric bioscavengers to remove OP toxins from circulation. FIG.1 is a schematic illustration of a stoichiometric bioscavenger showingthe stoichiometric binding and sequestering of an inhibitor by formationof an enzyme and inhibitor complex. As shown in FIG. 1, the formation ofa typical overall enzyme-inhibitor complex is illustrated as an enzyme Ereacting with an inhibitor I, to form the enzyme-inhibitor complex EI(as shown in FIG. 1 within a dashed circle). The double headed arrow ofFIG. 1 indicates that the reaction of the enzyme E and the inhibitor Ito form the enzyme-inhibitor complex EI occurs stoichiometrically inboth directions. Regeneration of the enzyme-inhibitor complex EI mayoccur by reacting the enzyme-inhibitor complex EI with a freeregenerating molecule R. As shown in FIG. 1, the regenerating molecule Rbonds to the inhibitor I of the enzyme-inhibitor complex EI removing theinhibitor I from the complex EI to form end products of a regeneratedenzyme E and a non-toxic inhibitor-regenerating molecule compound IR.The enzyme kinetics of the regeneration of the enzyme E is controlled bythe stoichiometric amounts of free regenerating molecule Randenzyme-inhibitor complex EI present. Enzymes from many sources with avariety of activities against OP compounds have been investigated aspotential regenerating bioscavengers. The development of mutated OPhydrolyzing enzymes as regenerating bioscavengers of nerve agents andpesticides, cloned either from bacterial sources (phosphotriesterases,PTEs) or non-human mammalian origin (serum paraoxonase 1, PON1), havedemonstrated significant progress in terms of their catalytic efficacy(kcat/Km) in hydrolyzing nerve agents. However, the versatility towarddifferent OP compounds (i.e. G- and V-agents) and immune-compatibilityhas to be further examined. While many of these OP hydrolases showpromise for decontamination of OP compounds, if OP compounds areconsidered to be pseudo-substrates of AChE, then none of the proposedenzymes has the substrate range of AChE. Pertinently, cholinesterasesare inhibited by all toxic OP pesticides and nerve agents and the onlydifferences between the various OP inhibitors are their kinetics of AChEand BChE inhibition.

Thus, the present investigation considers converting cholinesterasesfrom stoichiometric to regenerating bioscavengers to gain efficientdetoxification towards numerous OP compounds, both known and unknown(such as, G-agents, a family of agents named for their German inventors,and V nerve agents, a second family of agents believed to be named forviscosity, venomous, or victory as well as thio-OP and oxo-OPpesticides). In contrast, the development of OP hydrolase (OPH) (e.g.,OPH and PONI) usually requires tailoring the enzyme mutants towardspecific P—X bonds of OP compounds (where X is the leaving group, OR, SRor halogen). Moreover, G- and V-nerve agents are chiral compoundscontaining optical isomers with different stereochemistry ofsubstituents around the phosphorus atom. The Sp stereoisomer of G-agentssuch as sarin and cyclosarin and V-agents such as O-ethylS-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) is more toxicthan its Rp counterpart by 2-3 orders of magnitude. The naturallyoccurring PTE and PONI detoxify the less toxic Rp isomer more rapidlyand tremendous efforts have been made to convert the stereospecificityof PTE and PONI by genetic manipulations. Conversely, AChE and BChE areinhibited more rapidly by the Sp isomer of nerve agents. For instance,AChE is inhibited by Sp-VX 115-fold more rapidly than by Rp-VX, andtherefore both AChE and BChE could serve as native protein scavengersfor rapid detoxification of these toxic OP stereoisomers.

With the current tool set, a universal antitoxin would require a mixtureof enzymes to cover all contingencies. This consideration has led tomutational studies of native ChEs to develop enzymes capable ofdegrading a wide variety of OP compounds. These studies have producedengineered enzymes that have some catalytic activity toward multiple OPcompounds but their catalytic activities were limited. Millard C B,Lockridge 0, Broomfield C A., Organophosphorus acid anhydride hydrolaseactivity in human butyrylcholinesterase: synergy results in a somanase,Biochemistry, 37(1):237-47 (1998); Millard C B, Lockridge 0, BroomfieldC A., Design and expression of organophosphorus acid anhydride hydrolaseactivity in human butyrylcholinesterase, Biochemistry, 34(49):15925-33(1995).

Much of the work regarding stoichiometric bioscavengers has centered onthe use of human butyrylcholinesterase (HuBChE), an adventitious naturalbioscavenger found as soluble protein in human plasma. Bioscavengers aredesigned to lower the concentration of the toxin in the blood thuskeeping the OP compounds from reaching their AChE targets in theperipheral nervous system and the central nervous system. The key tousing bioscavengers like butyrylcholinesterase (BChE) is to maintainhigh serum concentrations and binding potential at effective levels.Native BChE concentration in plasma is about 50-80 nM, well below thatneeded to neutralize a lethal dose of OP. An effective dose of BChEagainst exposure to OP at several times the LD50 (defined as lethal dosethat kills half of the test animals under controlled conditions), wouldbe about 3 mg/kg iv (or 210 mg per 70 kg body weight). Processing all ofthe expired human plasma in U.S. blood banks or Red Cross stocks for anentire year would result in only about 5,000 such HuBChE doses. Clonedsources of HuBChE have been developed to increase production of theenzyme but the enzyme must be processed post-purification because ofsialylation differences in the producing cells adding to the cost.Transgenic goats which produce recombinant HuBChE in their milk havebeen under development for some time but display problems with lactationthat have delayed their use as an alternative source of HuBChE.

An improved alternative to a stoichiometric bioscavenger is aregenerating bioscavenger. FIG. 2 is a schematic illustration of a novelregenerating bioscavenger enzyme that cleaves the OP toxin substrateinto biologically inert molecules, or molecules that are chemicallynon-harmful to organisms, also known as non-toxic molecules. Thebioscavenger of FIG. 2 employs an enzyme E including covalently attachedregenerating molecules Rn, where n is more than one or a plurality ofregenerating molecules bound to the enzyme. Following reaction with aninhibitor I, the enzyme E and attached regenerating molecules Rn form acomplex with the inhibitor I (see FIG. 2 within the dashed-lineellipse). As illustrated, once the enzyme E/regenerating moleculesRn/inhibitor I complex is formed at least one regenerating molecule Rreacts with the inhibitor I to form the end products of a biologicallyinert inhibitor Io compound, and a regenerated bioscavenger enzyme Eincluding the regenerating molecules Rn. The initial hydrolysis of thefull OP followed cleavage of the phosphoryl from the active site serineresults two non-reactive molecules. Double arrows in FIG. 2 representthe rapid kinetics afforded by the multiple regenerating molecules atthe enzyme site which react instantaneously (i.e., at a rate equal to orapproximately equal to the rate of diffusion) with the inhibitor or OPmolecule inhibiting the active site to regenerate and detoxify theenzyme.

The present investigation considers a hybrid approach of coupling theadvantages of first and second generation oximes (which arepseudo-catalytic versus stoichiometric in that they generate a furtheroxime upon hydrolysis) with native and engineered esterases. Indeed,this approach has resulted in OP compound detoxification in solutiontogether with regeneration of the enzyme polyoxime conjugate describedherein. The degree of regenerative cycling is of course limited by thenumber of oximes available per enzyme molecule but all enzymes suffer instability during turnover even with natural substrates. However, asmentioned above, the short half-life of oximes in vivo limits the use offree oximes as unbound pseudo-cofactors for cholinesterases.

The present investigation describes a solution to the current state ofscavenging by using polymer-based protein engineering to conjugate oximecontaining polymers directly to esterases providing a tetheredpseudo-cofactor. Precisely engineered design of the polymer conjugatesand the advantages of controlled radical polymerization (such as ATRP)can provide the engineered esterase polymer conjugates protection fromimmune surveillance and extended serum residence times.

Polymer-Based Protein Engineering.

According to various aspects of the present invention, enzymes, and inparticular esterases such as the cholinesterases chymotrypsin may bemodified and immobilized to target chemical agents. It has been shownthat polymer binding to multiple sites on the enzyme surface, as isdescribed herein, does not significantly alter enzyme activity of theesterase. As shown in LeJeune, K. E., Frazier, D. S., Caranto, G. R.,Maxwell, D. M., Amitai, G., Russell, A. J. and Doctor, B. P., Covalentlinkage of mammalian cholinesterases within polyurethane foams, Proc.Med Def Biosc. Rev., vol. 1, 223-230 (1996), the nerve agent detectionsystem developed with the Agentase/FLIR® is based on multipoint bindingto amine groups of AChE to polyurethane foams. See also, U.S. Pat. Nos.6,759,220 and 6,291,200.

Most of the current techniques for polymer modification of proteinsdepend upon a “grafting to” approach where pre-formed polymers areconjugated to chemically reactive amino acid side chains.N-hydroxysuccinimide (NHS) chemistry is used to modify the availableamine groups primarily at surface available lysine residues. This hasbeen used in many previous enzyme modification studies most notably bymodifying chymotrypsin with polymers that contained radical sinks whichprotected the enzyme activity on TiO2 surfaces under intense UVillumination. These studies showed that polymer conjugation couldprovide additional functionality to enzymes. A possible drawback of“grafting to” conjugation is that not all available sites may beconjugated leading to a non-homogeneous product that may complicateanalysis and give batch dependent results.

In recent work, NHS chemistry was used to bind a specially designedpolymerization initiator, N-2-bromo-2-methylpropanoyl-J3-alanineN-hydroxysuccinimide ester to free amine groups on chymotrypsin inaqueous buffer followed by atom transfer radical polymerization (ATRP)which has recently been configured to include reactions that take placein fully aqueous conditions. See PCT/US2014/035033 filed 22 Apr. 2014;Murata H, Cummings C S, Koepsel R R, Russell A J, Polymer-Based ProteinEngineering Can Rationally Tune Enzyme Activity, pH-Dependence, andStability, Biomacromolecules, 10:14(6):1919-26 (2013); Cummings C,Murata H, Koepsel R, Russell A J, Tailoring enzyme activity andstability using polymer-based protein engineering, Biomaterials, 34:7437-7443 (2013); Matyjaszewski K., Atom Transfer RadicalPolymerization: From Mechanisms to Applications, Jsr J Chem.52(3-4):206-20 (2012); Matyjaszewski K., Atom Transfer RadicalPolymerization (ATRP): Current Status and Future Perspectives,Macromolecules, 45(10):4015-39 (2012), each incorporated herein byreference.

Using an initiator and ATRP under aqueous conditions densepoly-N,N-dimethylaminoethyl methacrylate (DMAEMA)-chymotrypsinconjugates were synthesized with relatively narrow molecular weightdistributions. The chymotrypsin-PDMAEMA conjugates had higher relativeenzyme activities compared to native chymotrypsin below pH 8. Indeed,the conjugates had a ten-fold higher enzyme activity than native enzymeat pH 5. Poly-DMAEMA (PDMAEMA) is a pH-responsive polymer with acondensed conformation above pH 8 and an open extended form at lower pH.With the polymer-enzyme conjugate, points of inflection in thepH-activity profiles were observed that coincided with points at whichthe molecular conformation of the conjugate changed. In the experimentsdescribed in PCT/US2014/035033, Murata H, et al., Biomacromolecules,(2013) (supra); and Cummings C, et al., Biomaterials, (2013) (supra),nearly saturated conjugation with 12 of 13 potential sites modified wasachieved. These results demonstrate that high density polymerconjugation is achievable and that conjugates using responsive polymerscan influence enzyme behavior. The active site residues of the serineprotease chymotrypsin are similar to those of the cholinesterases withthe mechanistically critical nucleophilic serine residue being the siteof inhibition in the cholinesterases.

The present invention advances the understanding of enzymatic mechanismsas well as polymer-oxime and polymer-enzyme interactions especiallythose involved in mitigating contamination by OP Chemical Warfare NerveAgents (CWNA). Converting ChE from a stoichiometric bioscavenger (i.e.,free ChE and/or free oximes) into a regenerating bioscavenger (i.e., ChEpolyoxime conjugate) may reduce significantly (1-2 orders of magnitude)the required protein dose of ChE for injection and thus provide areasonably inexpensive alternative to stoichiometric bioscavengers and alonger acting antitoxin than free oximes alone.

In various aspects, the present investigation describes the engineeringof a polymer-protein conjugate composed of a polymer or polymerscontaining oxime functionality with an enzyme from the esterase group.Enzymes from the esterase group may include cholinesterase,acetylcholinesterase, butyrylcholinesterase, and chymotrypsin.Alternative names for acetylcholinesterase are known to persons havingordinary skill in the art. For example, alternative names foracetylcholinesterase include RBC cholinesterase, erythrocytecholinesterase, serum cholinesterase, acetylcholine acetylhydrolase,acetylhydrolase, and forms of acetylcholinesterase encoded by the AChEgene(s), AChE, AChET, AChEH, and AChER. Alternative names forbutyrylcholinesterase are also known to persons having ordinary skill inthe art. For example, alternative names for butyrylcholinesteraseinclude BChE, BuChE, pseudocholinesterase, plasma cholinesterase, oracylcholine acylhydrolase.

In various aspects, the plurality of oxime functional groups of theesterase polymer conjugate composition comprises an aldoxime. Morespecifically, the aldoxime may comprise 2-pyridine aldoxime (2-PAM).Also known as Pralidoxime, or 2-pyridine aldoxime methyl chlorideusually as the chloride or methiodide salts is a member of the oximegroup of compounds that bind to organophosphate-inactivatedacetylcholinesterase. The 2-PAM aldoxime monomer has the ability toattach to an unblocked anionic site of the inhibitingacetylcholinesterase enzyme. The 2-PAM subsequently binds to theorganophosphate inhibiting molecule. The organophosphate once bound tothe 2-PAM oxime molecule changes conformation and is released from itsbinding to the acetylcholinesterase enzyme active site. The disjoined OPinhibitor/2-PAM oxime antidote then unbinds from the enzyme, which isnow able to function again.

In various aspects, the plurality of oxime functional groups maycomprise a ketoxime. A ketoxime is similar in structure to an aldoximeas both are of the oxime group of organic molecules. The general formulafor an oxime is R1R2C═N—O—H. In an aldoxime, the R1 group is an organicside-chain and R2 may be hydrogen, while in a ketoxime the R2 groupcomprises another organic functional group.

In various aspects, the plurality of oxime functional groups maycomprise at least one bis-pyridinium oxime. For example, the pluralityof oxime functional groups may comprise at least one of trimedoximebromide (TMB-4, also known as dipyroxime),1,1′1Methylenebis[4-[(hydroxyimino)methyl]-pyridinium dibromide (MMB-4),or combinations thereof.

In various aspects, the at least one polymer covalently conjugated tothe esterase forms a long lived covalent conjugate. The number ofpolymers covalently conjugated to the esterase may include from about80% to about 100% of the sites available for covalent attachment withinthe active site of the esterase. For example, the number of polymerscovalently conjugated to the esterase may include from about 85% toabout 95%, from about 90% to about 95%, or for any range subsumedtherein, For example, from about 84% to about 96% of the sites availablefor covalent attachment within the active site of the esterase.

In various aspects, the number of polymers covalently conjugated to theesterase may be limited by the number of lysine residues exposed to thesurface of the enzyme active site. In various aspects, the number ofpolymers may be bound to about 85%, about 90%, about 95%, or up to 100%of the surface lysine residues of the enzyme active site. Furtherpolymers may be bound to other surface residues of the enzyme. Invarious aspects, the number of polymers covalently conjugated to theesterase may be limited to a range of about 90% to about 95% for theesterase polymer conjugate to maintain a level of enzyme activitysubstantially similar to or substantially equal to that of a nativeenzyme. For example, it has been found that too many polymers bound tothe enzyme may diminish enzyme activity. Typically, as many as about 95%of the surface residues (e.g., lysine residues) may be attached to apolymer without loss of enzyme activity.

In various aspects, the covalent attachment of multiple polymers ontothe surface of the enzyme active site provides for delayed clearancefrom the body. For example, the long lived esterase polymer conjugatemay remain within circulation in the body of a mammalian recipientthereof for more than about one day, for more than about 5 days, formore than about 1 week, for more than about 2 weeks, for more than about3 weeks, or for more than about 1 month. In various aspects, the longlived covalent conjugate is maintained in the body for a time periodranging from more than about one day to less than about one week, frommore than about 3 days to less than about two weeks, from more thanabout one week to less than about three weeks, or for more than abouttwo weeks to less than about one month, or for more than about onemonth, for any sub-range subsumed therein, such as for more than about 2days to less than one week.

In various aspects, the at least one polymer may comprise at least oneenvironmentally responsive monomer. Environmentally responsive monomersmay change conformation, charge, or physical structure in response to astimulus in the proximate environment of the monomer. For example, andas shown in Table 3, the at least one polymer may comprise at least oneof poly(N-isopropylacrylamide), poly(oligo(ethylene glycol) methyl ethermethacrylate), poly(sulfobetaine methacrylate), poly(N,N,dimethylaminoethyl methacrylate), poly((meth)acrylate), or the like, orcombinations thereof.

In various aspects, the esterase polymer conjugate composition maycomprise at least one polymer exhibiting a polymer length ranging from aminimum of at least 2 monomer repeats to about 1000 monomer repeats. Forexample, the polymer length may range from a minimum of at least 5monomer repeats to about 750 monomer repeats, from a minimum of at least25 monomer repeats to about 500 monomer repeats, from a minimum of atleast 100 monomer repeats to about 250 monomer repeats, or for any rangesubsumed therein, For example, from a minimum of 10 monomer repeats toabout 900 monomer repeats. The only upper limit on the number or lengthof monomers is that number or length that will avoid hindering contactof the oxime functional group or groups sufficient to interact with theactive site of the enzyme for neutralization of the inhibiting moiety.

In various aspects, the esterase polymer conjugate composition maycomprise macromolecules composed of more than one monomeric repeatingunit, or co-polymers. In various aspects, the esterase polymer conjugatemay comprise at least one polymer that is a co-polymer comprising atleast two different monomers, wherein at least one monomer comprises amember selected from the group consisting of aldoximes, ketoximes,muco-adhesion monomers, polyethylene glycol, bis-pyridinium oximes,N,N-dimethylacrylamide, N-isopropylacrylamide, (meth)acrylate,N,N-dimethylaminoethyl methacrylate, carboxyl acrylamide,2-hydroxylethylmethacrylate, N-(2-hydroxypropyl)methacrylamide,quaternary ammonium monomers, sulfobetain methacrylate, oligo(ethyleneglycol) methyl ether methacrylate, 2-PAM monomers, 4-PAM monomers,Clickable azide monomers, and the like, and combinations thereof.

In addition, the co-polymer of the esterase polymer conjugate maycomprise at least two different monomers, wherein at least one monomermay comprise a varied topology from at least one different monomer ofthe co-polymer. More specifically, the varied topology of the at leastone monomer may include block, random, star, end-functional, or in-chainfunctional co-polymer topology. For example, at least one monomer of theco-polymer may include at least one monomer of a di-block topology. Theco-polymers, monomers for di-block formation, monomers including an endfunctional group, or in-chain functional co-polymers may be synthesizedutilizing the materials and methods described in U.S. Pat. No. 5,789,487to Matyjaszewski et al, U.S. Pat. No. 6,624,263 to Matyjaszewski et al,U.S. Patent Application Publication No. 2009/0171024 to Jakubowski etal., and Matyjaszewski, K, and Davis, T. P., ed., Handbook of RadicalPolymerization, John Wiley and Sons, Inc., Hoboken, N.J. (2002) areincorporated herein by reference in their entirety, the patents andpatent applications including their specifications, drawings, claims andabstracts.

In various aspects, the esterase polymer conjugate may include aplurality of polymers each covalently conjugated to the esterase, eachpolymer comprising a plurality of monomer units wherein at least onesaid monomer unit comprises an oxime functional group and wherein aplurality of monomer units of each polymer comprises an oxime functionalgroup. In various aspects, a plurality of monomer units of each polymerof the plurality of polymers may comprise an oxime functional group. Invarious aspects, the plurality of polymers may comprise co-polymerswherein each co-polymer may include at least two different monomers inwhich at least one monomer comprises a member selected from the groupconsisting of aldoximes, ketoximes, muco-adhesion monomers, polyethyleneglycol, bis-pyridinium oximes, N,N-dimethylacrylamide,N-isopropylacrylamide, (meth)acrylate, N,N-dimethylaminoethylmethacrylate, carboxyl acrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and the like, and combinations thereof.

Where the esterase polymer conjugate comprises a co-polymer, theco-polymer may comprise a member of the group consisting of astatistical co-polymer, a random co-polymer, an alternating co-polymer,a block co-polymer, a di-block co-polymer, a tri-block co-polymer, agraft co-polymer, a multiple-block co-polymer, or the like, orcombinations thereof.

In various aspects, the esterase polymer conjugate may comprise aplurality of polymers each covalently conjugated to the esterase andeach polymer may comprise a plurality of monomer units wherein at leastone said monomer unit comprises an oxime functional group and theplurality of polymers comprises a plurality of co-polymers and aplurality of homopolymers. Further, each co-polymer of the plurality ofco-polymers may comprise at least two different monomers, wherein atleast one monomer comprises a member selected from the group consistingof aldoximes, ketoximes, muco-adhesion monomers, polyethylene glycol,bis-pyridinium oximes, N,N-dimethylacrylamide, N-isopropylacrylamide,(meth)acrylate, N,N-dimethylaminoethyl methacrylate, carboxylacrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and the like, and combinations thereof. In addition, each homopolymer ofthe plurality of homopolymers comprises a member selected from the groupconsisting of aldoximes, ketoximes, muco-adhesion monomers, polyethyleneglycol, bis-pyridinium oximes, N,N-dimethylacrylamide,N-isopropylacrylamide, (meth)acrylate, N,N-dimethylaminoethylmethacrylate, carboxyl acrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, Clickable azide monomers,and the like, and combinations thereof.

In various aspects, a composition may comprise a bioconjugatecomposition formed through a stable covalent link between twobiomolecules. The bioconjugate composition may comprise an esterase andat least one polymer covalently conjugated to the esterase. The esteraseof the bioconjugate composition may comprise any of the esterases (e.g.,acetylcholinesterase, cholinesterase, etc.) described for the esterasepolymer composition as described herein. In various aspects, theesterase of the bioconjugate composition may comprise chymotrypsin. Thebioconjugate composition may further comprise at least one polymercovalently conjugated to the esterase. The at least one polymer maycomprise a plurality of oxime functional groups and at least one oximefunctional group of the plurality of oxime functional groups ispositioned to react, in use, with a phosphoryl functional group when aninhibitor having a phosphoryl functional group attaches to an activesite of the esterase.

In various aspects, the bioconjugate composition may include at leastone polymer. For example, the bioconjugate composition may comprise atleast one polymer comprising a flexibility sufficient to react, in use,with a phosphoryl functional group when an inhibitor having a phosphorylfunctional group attaches to the active site of the esterase. In variousembodiments, the bioconjugate composition may include any of thepolymers disclosed herein.

In various aspects, the bioconjugate composition may include functionalgroups. More specifically, the bioconjugate composition may include atleast one oxime functional group selected from any of the oximefunctional groups described herein.

In various aspects, the bioconjugate composition may include at leastone monomer. Specifically, the bioconjugate composition monomer mayinclude any of the monomers described herein. For example, thebioconjugate composition may include at least one of any of theenvironmentally responsive monomers as described herein.

In various aspects, the bioconjugate composition may serve as a drugdelivery system. More specifically the bioconjugate composition maycomprise a drug delivery system for polymeric antitoxins. For example,the bioconjugate composition may protect polymeric antitoxins such aspolyoximes from clearance from the body and degradation forming a longlived covalent conjugate that is maintained in the body for time periodsthe same or similar to those described for the esterase polymerconjugate.

In various aspects, the bioconjugate composition may include at leastone polymer, the at least one polymer including a polymer length that isthe same or similar to that described with respect to the esterasepolymer conjugate.

In various aspects, the at least one polymer of the bioconjugatecomposition may comprise a co-polymer. For example, the at least onepolymer may include at least two different monomers as described herein.In various aspects, the bioconjugate composition may include a pluralityof polymers each covalently conjugated to the esterase. Morespecifically, each polymer may include a plurality of monomer units ormonomers as described herein. In various aspects, the plurality ofpolymers may include co-polymers. For example, the co-polymers mayinclude at least two different monomers as described herein.

In various aspects, the plurality of polymers may include a plurality ofco-polymers and a plurality of homopolymers. More specifically, eachco-polymer and each homopolymer may include at least one of the monomersas described herein.

In various aspects, the bioconjugate composition may interact with andtherefore at certain times as it functions, the bioconjugate compositionmay be bound to an inhibitor. For example, the bioconjugate compositionmay interact with and include VX, Sarin, Soman, DFP, Paraoxon,Parathion, or the like, or combinations.

In various aspects, a method is provided including administering abioscavenger to an individual suffering from toxin exposure. Morespecifically, the toxin exposure may be due to exposure of theindividual to an organophosphate toxin, a nerve agent, a neurotoxin. Thebioscavenger may include at least one polymer attached to an enzyme. Forexample, the at least one polymer may include a plurality of oximegroups as described herein covalently bound to an esterase. In variousaspects, upon exposure of the bioscavenger to an OP toxin the methodincludes reacting at least one of a plurality of oxime functional groupswith at least one covalently inhibited residue of an esterase todetoxify and regenerate the bioscavenger.

In various aspects, the method may include administering thebioscavenger to an individual person or to a mammalian individual.

In various aspects, the plurality of oxime functional groups of thebioscavenger conjugate may include an oxime functional group positionedto exert a nucleophilic attack on an inhibitor, when in use, theinhibitor attaches to an active site of the esterase. For example, themethod may include an oxime functional group positioned to exert anucleophilic attack on a phosphoryl functional group inhibiting theactive site of the esterase, resulting in the removal of the phosphorylfunctional group from the active site.

In various aspects, the method of administering a bioscavenger to anindividual suffering from OP toxin exposure may include administering along lived covalent enzyme polymer conjugate that is maintained in thebody for time periods the same or similar to those described for theesterase polymer conjugate.

In various aspects, the esterase of the bioscavenger conjugate maycomprise any of the esterases described for the esterase polymercomposition as described herein. For example, the esterase of thebioscavenger conjugate may comprise chymotrypsin, acetylcholinesterase,cholinesterase, etc.

In various aspects, the administered bioscavenger may include aplurality of oxime functional groups. For example, the administeredbioscavenger may include at least one oxime functional group selectedfrom any of the oxime functional groups described herein. In variousaspects, the administered bioscavenger may include at least one polymer,the at least one polymer including a polymer length that is the same orsimilar to that described with respect to the esterase polymerconjugate.

In various aspects, the administered bioscavenger may include at leastone monomer. Specifically, the bioconjugate composition monomer mayinclude any of the monomers described herein. For example, theadministered bioscavenger may include at least one of any of theenvironmentally responsive monomers as described herein.

In various aspects, the at least one polymer of the administeredbioscavenger may comprise a co-polymer. For example, the at least onepolymer may include at least two different monomers as described herein.In various aspects, the administered bioscavenger may include aplurality of polymers each covalently conjugated to the esterase. Morespecifically, each polymer may include a plurality of monomer units ormonomers as described herein.

In various aspects, the plurality of polymers may include co-polymers.For example, the co-polymers may include at least two different monomersas described herein. In various aspects, the plurality of polymers mayinclude a plurality of co-polymers and a plurality of homopolymers. Morespecifically, each co-polymer and each homopolymer may include at leastone of the monomers as described herein.

In various aspects, a bioscavenger may comprise at least one polymercovalently conjugated to an esterase. The esterase of the bioscavengermay comprise any of the esterases (e.g., acetylcholinesterase,cholinesterase, etc.) described for the esterase polymer composition asdescribed herein. In various aspects, the esterase of the bioscavengermay comprise chymotrypsin.

In various aspects, the bioscavenger may comprise at least one polymercovalently conjugated to the esterase. The at least one polymer maycomprise a plurality of oxime functional groups and at least one oximefunctional group of the plurality of oxime functional groups ispositioned to exert a nucleophilic attack on a phosphoryl functionalgroup when, in use, a phosphoryl functional group is covalently attachedto an active site of the esterase effecting removal of the phosphorylfunctional group from the active site and regeneration of thebioscavenger. In various aspects, the at least one oxime functionalgroup comprises an oxime functional group positioned to exert anucleophilic attack on a phosphoryl functional group when, in use, aninhibitor having a phosphoryl functional group attaches to an activesite of the esterase. More specifically the nucleophilic attack resultsin the removal of the phosphoryl functional group from the active site.

In various aspects, the bioscavenger may serve as a drug deliverysystem. More specifically the bioconjugate composition may comprise adrug delivery system for polymeric antitoxins. For example, thebioscavenger may protect polymeric antitoxins such as oximes fromclearance from the body and degradation forming a long lived covalentconjugate that is maintained in the body for time periods the same orsimilar to those described for the esterase polymer conjugate.

In various aspects, the bioscavenger may include functional groups. Morespecifically, the bioscavenger may include at least one oxime functionalgroup selected from any of the oxime functional groups described herein.

In various aspects, the bioscavenger may include at least one polymer,the at least one polymer including a polymer length that is the same orsimilar to that described with respect to the esterase polymerconjugate.

In various aspects, the bioscavenger may include at least one monomer.Specifically, the bioscavenger monomer may include any of the monomersdescribed herein. For example, the bioscavenger may include at least oneof any of the environmentally responsive monomers as described herein.

In various aspects, the at least one polymer of the bioscavenger maycomprise a co-polymer. For example, the at least one polymer may includeat least two different monomers as described herein. In various aspects,the bioconjugate composition may include a plurality of polymers eachcovalently conjugated to the esterase. More specifically, each polymermay include a plurality of monomer units or monomers as describedherein. In various aspects, the plurality of polymers may includeco-polymers. For example, the co-polymers may include at least twodifferent monomers as described herein. In various aspects, theplurality of polymers may include a plurality of co-polymers and aplurality of homopolymers. More specifically, each co-polymer and eachhomopolymer may include at least two different monomers as describedherein.

The result of this investigation is a broad spectrum antidote that canbe used as an internal therapeutic for OP toxins primarily frompesticides and chemical weapons. The work of engineering an optimizedpolymer-protein conjugate uses the AChE-poly-2-PAM conjugate describedabove as the starting point.

Example 1

Oxime Polymer Synthesis

The synthesis of an acrylate polymer 3,4-dimethoxy-N-methylamphetamine(DMMA) 4-pyridine aldoxime (4-PAM) random copolymer as part of amulticomponent electrospun polyurethane material has been reported.Amitai G, Murata H, Andersen J D, Koepsel R R, Russell A J.,Decontamination of chemical and biological warfare agents with a singlemulti-functional material, Biomaterials, 31(15):4417-25 (2010). Thiswater soluble 4-PAM co-polymer demonstrated a dose-response pH dependentdetoxification of diisopropyl fluorophosphate (DFP). Thehydroxyl-ethyl-4-PAM residue was bound to the co-polymer backbone by anester bond allowing its controlled release from the polymer. Thereleased hydroxyl-ethyl-4-PAM was shown to reactivate DFP inhibited AChEat a similar rate as the antidote oxime 4-PAM. The same syntheticprocess was used to synthesize a 2-PAM polymer (see FIGS. 3A-3E). Thepolymer was synthesized by thermo polymerization as a random co-polymerof dimethyl acrylamide (DMAA) and methacrylbromide (MA-Br) (see FIG.3A). The polymer was then quaternized with 2-pyridine aldoxime (see FIG.3C) and modified to contain a terminal NHS moiety (see FIG. 3E).

FIG. 3A illustrates a schematic of the preparation of a brominecontaining polymer the first step in the synthesis of the firstAChE-polymer conjugate. Preparation of the bromine containing polymerincluded the following materials and methods:

Monomers DMAA (4.0 mL, 38.8 mmol), and 2-bromoethyl methacrylate (750mg, 3.9 mmol) were placed in a polymerization tube and covered with4,4′-azobis(4-cyanovaleric acid) (140 mg, 0.5 mmol) as an initiator and50 mL of Toluene. The monomer solutions were degassed by fivefreeze-pump-thaw cycles and then heated to 60° C. for 3 h. The resultingmixture was precipitated with diethyl ether (500 mL). Theether-insoluble part was filtrated off and dried overnight in vacuo:yield 2.8 g (73%), number average molecular weights (Mn) and thedistributions (Mw/Mn) of the obtained polymer was estimated by gelpermeation chromatography (GPC) on a Waters 600E Series with a dataprocessor, equipped with three polystyrene columns (Waters styragel HR1,HR2 and HR4), using DMF with LiBr (50 mM) as an eluent at a flow rate of1.0 mL/min, polymethylmethacrylate calibration, and a refractive index(RI) detector resulting in a Mn of 29,400 g/mol and the distributions of(Mw/Mn) 3.99. The chemical structure and concentration of bromine groupsof the obtained polymer was determined by ¹H NMR spectra (see FIG. 3B),which was recorded on a Bruker Avance (300 MHz) spectrometer in DMSO-d6.When this polymer was grafted to AChE the resulting conjugate had anaverage of 6 of the 13 accessible lysines conjugated to the enzyme. ThisAChE poly-2-PAM covalent conjugate was designed to demonstrate theeffect of oximes conjugated to the enzyme surface.

FIG. 3C is the chemical reaction of the quaternization reaction with2-PAM. The quaternization reaction with 2-PAM procedure included thefollowing materials and methods: The bromine containing polymer of FIGS.3A and 3B (2.9 g, 2.1 mmol of bromine), syn-2-pyridinealdoxime (650 mg,5.3 mmol) and toluene were placed into a flask and refluxed at 120° C.overnight. After cooling down, the mixture was precipitated in diethylether and filtered. The product was purified by decantation withacetone/diethyl ether several times. The obtained polymer was dried invacuo: yield 2.5 g, the number average molecular weights (Mn) and thedistributions (Mw/Mn) of the obtained polymer was estimated by GPCresulting in a Mn of 34,400 and the distributions of (Mw/Mn) 4.30. Thechemical structure and concentration of 2-PAM groups of the obtainedpolymer was determined by ¹H NMR spectra as shown in FIG. 3D.

FIG. 3E illustrates the chemical generation of an N-oxysuccinimide estergroup on the 2-PAM polymer shown in FIGS. 3C and 3D. Preparation of thegeneration of N-oxysuccinimide ester group on the 2-PAM polymer includedthe following materials and methods: Ethyl(dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) (39 mg, 0.2 mmol) andN-hydroxysuccinimide (NHS) (24 mg, 0.2 mmol) were added in a solution ofthe 2-PAM polymer (680 mg, 0.02 mmol of —COOH end group) in deionizedwater (20 mL) and stirred at room temperature for 30 min. The obtainedpolymer was isolated by dialysis using a molecular weight cut off 1,000dialysis tube in the refrigerator (4° C.), then lyophilized.

AChE-Polymer Conjugates

An AChE-polymer conjugate was synthesized including the 2-PAM containingpolymer as shown in FIG. 3E synthesized using a “grafted to” approach(see FIG. 3F). FIG. 3F illustrates a schematic of the conjugation of the2-PAM polymer shown in FIG. 3E with AChE. Preparation of the conjugationof the 2-PAM polymer with AChE included the following materials andmethods: Polymer from the generation of the N-oxysuccinimide ester groupon the 2-PAM group of FIG. 3E (210 mg, 6.2 μmol) was added in a solutionof AChE (from Electrophorus electricus (electric eel), 10 mg, 3.1 μmolof amine groups) in 100 mM sodium phosphate buffer (10 mL, pH 8.0) andstirred at 4° C. overnight. The obtained AChE-2-PAM conjugate wasisolated by dialysis using a molecular weight cut off 50 kDa dialysistube in the refrigerator (4° C.), overnight, then lyophilized. 10 wt %of AChE in the conjugate was found by bicinchoninic acid (BCA) proteinassay and the polymer grafting density was determined by fluorescamineamine assay, respectively.

A BCA protein assay was used to determine the protein percentcomposition of AChE within the AChE-2-PAM conjugate using the followingmaterials and methods: Solutions (25 μL each) of varying concentrationsof native AChE (0.05-1.0 mg/mL; used for preparation of a standardcurve) and 1.0 mg/mL of AChE-2-PAM polymer conjugate in deionized waterwere mixed with 1 mL of BCA solution (15 mL of Bicinchoninic Acidsolution (Sigma-Aldrich) with 300 μL, of Copper (II) sulfate solution(Sigma-Aldrich)). The solution was incubated at 60° C. for 15 min.Absorbance of the solution at 562 nm was measured by UV/VISspectrometer. Protein concentration in the conjugate was estimated bycomparison to native AChE standards.

A fluorescamine amine assay was used to determine the number ofinitiators or polymers attached to the enzyme surface usingfluorescamine. In a fluorescamine amine assay the fluorescamine moleculereacts with primary amines (lysines) to form a fluorophore with anexcitation of 365 nm and an emission of 470 nm (see below). Equal molarsolutions (0.1 M phosphate buffer pH 8) of native enzyme and modifiedenzyme (at concentrations approximating 1 mg/ml) were prepared.Fluorescamine was added in dimethyl sulfoxide (DMSO) to 1 mg/ml finalconcentration. The sample was incubated for 15 minutes at 25° C. andfluorescence was measured at 470 nm.

Example 2

A second AChE-polymer conjugate was synthesized including a sulfonatemonomer using the “grafted from” approach as shown in FIGS. 4A and 4B.The AChE-polymer conjugate was synthesized directly from the surface ofAChE by ATRP including a sulfonate monomer. The synthesis of the secondAChE-polymer conjugate included the following materials and methods:

AChE (from Electrophorus electricus (electric eel), 23 mg, 7.2 μmol ofamine groups) was dissolved in 100 mM sodium phosphate buffer (20 mL, pH8.0) at 0° C. After adding N-2-bromo-2-methylpropionyl-β-alanineN-oxysuccinimide ester (7 mg, 20 μmol), the mixture was stirred in arefrigerator (4° C.) for 3 h and the AChE-initiator conjugate wasisolated by dialysis using a 50 kDa molecular weight cut off dialysistube in deionized water in a refrigerator (4° C.) for 24 h and thenlyophilized. 60% of the AChE surface lysines was reacted with the NHSfunctionalized ATRP initiator, which was estimated by TNBSA(2,4,6-Trinitrobenzene sulfonic acid) amine assay. To determine thepercent of AChE surface lysines reacted with the NHS functionalized ATRPinitiator the TNBSA amine assay was performed using the followingmaterials and methods:

Solutions (500 μLeach) of varying concentrations of native AChE (0.1-1.0mg/mL; used for preparation of a standard curve) and 1.0 mg/mL ofAChE-ATRP initiator conjugate in 100 mM sodium phosphate buffer (pH 8.5)were mixed with 250 μL of TNBS solution (20 μI, of 5% TNBS stocksolution (Sigma-Aldrich) with 10 mL of 100 mM sodium phosphate buffer(pH8.5). The solution was incubated at 37° C. for 2 h. 125 μL of 1 N HClaq. and 250 μL of water were added to each sample to stop and stabilizethe reaction. The absorbance of the solution at 345 nm was measured byultraviolet-visible spectroscopy using polymethyl methacrylate (PMMA)cuvettes. Concentration of unreacted primary amines on the AChE-ATRPinitiator conjugates was estimated by comparison to native AChEstandards.

As shown in the schematic of FIG. 4A, a solution of 3-sulfopropylmethacrylate potassium salt (22 mg, 89 μmop and AChE-initiator conjugate(5 mg, 0.89 μmol of initiator groups) in 50 mM sodium phosphate buffer(10 mL, pH 7.4) was sealed and bubbled with nitrogen for 50 min.Deoxygenated catalyst solutions of HMTETA(1,1,4,7,10,10-Hexamethyltriethylenetetramine, 1.0 μL, 3.6 μmol) andCu(I)Br (0.6 mg, 3.6 μmop in deionized water (1 mL) was then added tothe conjugation reactor under nitrogen bubbling. The mixture was sealedand stirred in a refrigerator (4° C.) for 18 h. AChE-sulfonate polymerconjugate was isolated by dialysis with a 50 kDa molecular weight cutoff dialysis tube in deionized water in a refrigerator (4° C.) for 24 hand then lyophilized. Molecular weight of the AChE-sulfonate polymerconjugate (146 kDa) was estimated by BCA protein assay using thematerials and methods described herein.

The AChE-poly-sulfonate conjugate was designed to assess the effect ofnegative charges on the surface of the enzyme. The synthesizedAChE-poly-sulfonate conjugate of FIGS. 4A and 4B had polymer grafted toall 13 available amine groups. The ¹H NMR spectrum of theAChE-poly-sulfonate conjugate is shown in FIG. 4B.

Activity assays of the AChE-poly-2-PAM conjugate and theAChE-poly-sulfonate conjugate showed that the esterase polyoximecovalent conjugate retained upward of 90% of the native activity whilethe AChE-poly-sulfonate conjugate retained less than 1%. An activityassay was completed on native AChE and the AChE-poly-2-PAM conjugateusing the following materials and methods: Native AChE andAChE-poly-2-PAM conjugate (0.4 μM each) were incubated in 1 μM paraoxonuntil completely inhibited (Native AChE incubated for 10 min, and theAChE-poly-2-PAM conjugate incubated for 15 min). The enzymes werediluted 40 fold into activity buffer (50 mM sodium phosphate pH 7.4, 1mM acetylthiocholine iodide and 0.74 mM 5,5′-Dithiobis (2-nitrobenzoicacid). Aliquots were tested for activity at the times indicated in thegraph of the data as shown in FIG. 5. It is likely that the negativelycharged shell generated around the AChE-poly-sulfonate conjugated enzymewas binding the positively charged substrate and limiting access to theactive site. It is unlikely, however, that there was a critical lysinethat remained unbound as the enzyme-initiator complex retained nativeactivity levels. The activity assay was also completed on native AChEand the AChE-poly-sulfonate conjugate using the same materials andmethods as described above for the native AChE.

Inhibition/reactivation assays were also performed. To achieve enzymeinhibition, the native AChE and the AChE-poly-2-PAM (0.4 μM each) wereincubated in 1 μM paraoxon until completely inhibited as described abovein the activity assay. Incubation time was 10 minutes for the nativeAChE and 15 min for the AChE-poly-2-PAM. The enzymes were diluted 40fold into an activity buffer. Reactivation assays were performed usingthe following materials and methods: The native AChE and the 2-PAMconjugate (3.6×10⁻⁷ M) were inhibited with Paraoxon (1×10⁻⁶ M). Aliquotswere sampled out into activity assay buffer containing 50 mM sodiumphosphate pH 7.4, 1 mM acetylthiocholine iodide and 0.74 mM 5,5′-Dithiobis (2-nitrobenzoic acid), and the change in optical density at412 nm (OD412)/sec was monitored for a decrease in the OD/sec to achieve90-95% inhibition. The inhibited enzyme was then diluted 1:50 intobuffer. Aliquots were sampled out into activity assay buffer. The rateof hydrolysis of substrate was monitored by the increase in absorptionat 412 nm.

The behavior of the AChE-poly-sulfonate conjugate in these assays wasunremarkable, closely mimicking native enzyme, yet another indicationthat the conjugation does not modify the enzyme active site. Incontrast, the esterase polymer covalent conjugate, AChE poly-2-PAM,showed a five-fold protection from acute inhibition at high paraoxonconcentration as a result of delivering the poly-2-PAM polyoxime withthe enzyme (see FIG. 5). More importantly, when the inhibitedAChE-poly-2-PAM polyoxime conjugate was diluted into buffer it underwentself-reactivation while the native enzyme remained completely inhibited.The rapid initial rate of self-reactivation is similar to that seen whennative enzyme is regenerated by free 2-PAM (as discussed in the recentwork by Amitai G, Murata H, Andersen J, Koepsel R, Russell A J,Decontamination of chemical and biological warfare agents with a singlemultifunctional material, Biomaterials, 31:4417-4425 (2010). Nativeenzyme does not self-reactivate or self-regenerate.

The steady state (see FIG. 5) reached after the initial reactivation islikely due to residual paraoxon carried over from the inhibitionreaction. These results suggest that the polymeric 2-PAM attached to theesterase enzyme is indeed functioning to self-regenerate esteraseactivity as intended. The ability of the esterase polymer covalentconjugate, AChE poly-2-PAM, to self-regenerate implies that at least aportion of the oxime residues of the surface attached polymers haveaccess to the active site of the enzyme.

Optimization of the esterase polymer covalent conjugate can includeoptimization of parameters such as the polymer length, monomer content,the length of the oxime tether, the oxime concentration, and the activeoxime identity that can result in a super-scavenger that is very likelycatalytic. Suitable local oxime concentrations as effective at theactive site of the enzyme may range between about 5 to about 500 oximefunctional groups per active site. For example, local oximeconcentrations as effective at the active site of the enzyme may rangefrom between about 1 to about 5 oxime functional groups per active site,about 1 to about 10 oxime functional groups per active site, about 5 toabout 10 oxime functional groups per active site, about 10 to about 25oxime functional groups per active site, about 25 to about 100 oximefunctional groups per active site, about 100 to about 500 oximefunctional groups per active site, or less than about 500 oximefunctional groups per active site. Actual oxime concentration per unitvolume may range from about 20 mM to about 0.0001 mM, from about 10 mMto about 0.001 mM, from about 5 mM to about 0.01 mM, or from about 1 mMto about 0.1 mM, or for any range subsumed therein, for example, fromabout 6 mM to about 0.006 mM.

Example 3

Esterase-Polyoxime Conjugate Synthesis Using ATRP

“Grafting from” ATRP has been used to synthesize enzyme-polymerconjugates Murata H, Cummings C S, Koepsel R R, Russell A J,Polymer-Based Protein Engineering Can Rationally Tune Enzyme Activity,pH-Dependence, and Stability, Biomacromolecules, 10:14(6):1919-26(2013); Cummings C, Murata H, Koepsel R, Russell A J, Tailoring enzymeactivity and stability using polymer-based protein engineering,Biomaterials, 34: 7437-7443 (2013).

A “grafting from” ATRP synthesis of an AChE-PDMAA/poly-2-PAM esterasepoly-oxime conjugate was completed as schematically represented in FIGS.6A-6D using the following materials and methods: AChE (fromElectrophorus electricus (electric eel), 20 mg, 6.3 μmol of aminegroups) was dissolved in 100 mM sodium phosphate buffer (20 mL, pH 8.0)at 0° C. After adding the N-2-cholopropionyl-β-alanine N-oxysuccinimideester (5.3 mg, 19 μmol), the mixture was stirred in a refrigerator (4°C.) for 3 hand the AChE-initiator conjugate was isolated by dialysisusing a 50 kDa molecular weight cut off dialysis tube in deionized waterin a refrigerator (4° C.) for 24 hand then lyophilized. 76% of the AChEsurface lysines were reacted with the NHS functionalized ATRP initiator,which was estimated by fluorescamine amine assay as described herein.

As illustrated in the schematic of FIG. 6A, a solution of DMAA(N,N-dimethyl acrylamide, 48 μL, 450 μmol), N-3-azidopropyl acrylamide(24 mg, 150 μmop and AChE-initiator conjugate (10 mg, 2.3 μmol ofinitiator groups) in deionized water was sealed and bubbled withnitrogen for 50 min. Deoxygenated catalyst solutions of Me₆TREN(Tris[2-(dimethylamino)ethyl]amine, 1.0 μL, 15 μmol) and Cu(I)Cl (1.5mg, 15 μmol) in deionized water (1 mL) was then added to the conjugationreactor under nitrogen bubbling. The mixture was sealed and stirred in arefrigerator (4° C.) for 18 h. AChE-PDMAA/Azide conjugate was isolatedby dialysis with a 50 kDa molecular weight cut off dialysis tube indeionized water in a refrigerator (4° C.) for 24 hand then lyophilized.Molecular weight of the AChE-PDMAA/Azide conjugate (350 kDa) wasobtained by BCA protein assay using the BCA materials and methodsdescribed herein. The chemical structure of AChE-PDMAA/Azide conjugatein D20 was determined by ¹H NMR spectrum (see FIG. 6B). Thirty-eightazide groups per grafted polymer chain, (i.e. 730 azide groups on singleconjugate molecules) were estimated by ¹H NMR spectrum.

As illustrated in the schematic of FIG. 6C, immobilization of 2-PAMgroups to the AChE-PDMAA/Azide conjugate of FIGS. 6A and 6B was carriedout by “Click” cycloaddition of alkyne 2-PAM in the presence of a coppercatalyst using the following materials and methods: A solution of theAChE-PDMAA/Azide conjugate of FIGS. 6A and 6B (16.2 mg, 33 μmol of azidegroups) in 25 mM potassium phosphate buffer (10 mL, pH 7.5) was sealedand bubbled with nitrogen for 50 min. Deoxygenated solution ofN-(3-butynyl)-2-pyridinealdoxime (17 mg, 66 μmol), CuSO4 (17 mg, 66μmol), HMTETA (18 μL, 66 μmop and ascorbic acid (12 mg, 66 μmol) indeionized water (1 mL) was then added to the conjugate solution undernitrogen bubbling. The mixture was sealed and stirred in a refrigerator(4° C.) for 18 h. AChE-PDMAA/2-PAM conjugate was isolated by dialysiswith a 50 kDa molecular weight cut off dialysis tube in deionized waterin a refrigerator (4° C.) for 24 h and then lyophilized. The chemicalstructure of obtained AChE-PDMAA/2-PAM conjugate was assigned by NMRspectrum (see FIG. 6D). Molecular weight of AChE-PDMAA/2-PAM conjugate(450.8 kDa) was determined by ¹H NMR spectrum. ¹H NMR was used todetermine that 50% of the azide groups were bound with alkyne 2-PAM by“Click” cycloaddition. The polymer components of the conjugates werefound to have narrow size distribution profiles with lengths governed bythe length of the polymerization reaction and monomer content controlledby the relative concentration. It was also found that the reaction couldbe stopped and resumed with different monomers allowing the generationof well-defined di-block co-polymers. To use ATRP to synthesize thepolymers, it may be helpful to change the method of attachment of theoxime groups to the polymer chain. In the synthesis of theAChE-poly-2-PAM, it was found that acrylate derivatives of PAM acted aschain terminators in ATRP and their attachment by quaternizationrequired high temperature, making it less suitable for the enzyme. Onechosen way to get around both of these problems is to use ATRP withacrylate monomers with terminal azides and oximes that are synthesizedwith an alkyne and then added to the polymer through “Click chemistry”(see Table 1).

The terms “Click chemistry,” “Click cycloaddition,” “Clickable,” and“Click reactions” refer to chemical reactions that are high yielding,wide in scope, create only by products that can be removed withoutchromatography, are stereospecific, simple to perform, and can beconducted in easily removable or benign solvents. Vyas, S., Hadad CM,Reactivation of model cholinesterases by oximes and intermediatephosphyloximes: A computational study, Chem Biol Interact, 175(1-3):187-191 (2008), which is incorporated in its entirety by reference.Several types of reactions have been identified that fulfill thesecriteria, thermodynamically-favored reactions that lead specifically toone product such as nucleophilic ring opening reactions of epoxides andaziridines, non-aldol type carbonyl reactions, such as the formation ofhydrazones and heterocycles, additions to carbon-carbon multiple bonds,such as oxidative formation of epoxides and Michael Additions, andcycloaddition reactions. Because of the versatility and selectivity ofthe “Click” reaction a variety of protein polymer conjugates can bedesigned. These include: polymers with different oxime groups; blockcopolymers with oxime and environmentally responsive blocks; randomco-polymers of chain extender and oxime monomers; polymers with oximemonomers with different length tethers to the polymer backbone; andmixtures of homopolymers. Synthesis of the enzyme-polyoxime conjugatesare based on AChE with the inclusion of BChE or other esterase,cholinesterase, or combinations thereof, as an optimization alternative.Alkyne-2-PAM derivatives for “Click” cycloaddition to azide groups onthe AChE-polymer conjugate can be prepared by varied synthetic pathwaysusing quaternization, esterification or condensation reaction of2-pyridinecarboxyaldehyde and alkyne derivatives as shown in Table 1.

TABLE 1 Preparation of “Click” PAM monomers Reactant Synthesis Product

As shown in Table 1, N-(3-butynyl)-2-pyridine aldoxime was synthesizedby quaternization of syn-2-pyridinealdoxime and 4-bromo-1-butyne.Synthesis of N-(3-butynyl)-2-pyridine aldoxime includes the followingmaterials and methods: 4-bromo-1-butyne (1.3 g, 10 mmol) was added to asolution of syn-2-pyridinealdoxime (1.0 g, 8.0 mmol) in acetonitrile(100 mL) and refluxed at 100° C. overnight. After cooling down thesolution to room temperature, the product was precipitated into diethylether. The obtained compound was dried in vacuo: yield 250 mg, ¹H NMR(spectra shown in FIG. 6E) (300 MHz, DMSO-d6) δ 2.87 (t, 2H, J=6.6 Hz,NCH₂CH₂C≡CH), 3.07 (s, 1H, NCH₂CH₂C≡CH), 4.93 (t, 2H, J=6.6 Hz,NCH₂CH₂C≡CH), 8.14, 8.43, 8.57 and 9.05 (4H, pyridine ring), 8.83 (s,1H, —CH═NOH), and 13.17 (broad s, 1H, —CH═NOH) ppm.

In a series of optimization rounds of the AChE-poly-2-PAM conjugate, thefirst round of the optimization process may use derivatives of 2-PAMbound covalently to the polymer backbone by spacers of various chainlengths. Synthesis of other alkyne derivatives of 2-PAM exhibitingvarying linkages and alkyl groups (see Table 1) may be achieved usingmaterials and methods known to persons of ordinary skill in the art. Invarious aspects, other suitable alkyne derivatives of 2-PAM for “Click”cycloaddition to azide groups on the AChE-polymer conjugate are shown inTable 2.

TABLE 2 Alkyne derivatives of 2-pyridine aldoxime Structure Substituents

In various aspects, other rounds of optimization of the AChE-poly-2-PAMconjugate may use derivatives of bis-pyridinium oximes. For example,trimedoxime bromide (TMB-4) or1,1-methylenebis[4-[(hydroxyimino)methyl]-pyridinium] dimethanesulfonate(MMB-4), and the ketoximes may be used to optimize the structure of theesterase polymer conjugate. (Radie Z et al., Refinement of StructuralLeads for Centrally Acting Oxime Reactivators of PhosphylatedCholinesterases, J. Biol. Chem., 287: 11798-11809 (2012). It ispertinent to note that ketoxime analogues of 2-PAM aldoxime generateoximes after their nucleophilic attack on the phosphoryl-ChE conjugatesduring reactivation of the enzyme. Thus, polymers containing quaternarypyridinium ketoximes (e.g., phenyl or methyl 2-Pyridinium aldoximemethochloride) were also tested as reactivators that could enhance theability of the polymer-engineered ChE into a true pseudo-catalytic OPhydrolase. Kitz, R J, Ginsburg S, Wilson I B, Activity-structurerelationships in reactivation of diethylphosphoryl acetylcholinesteraseby phenyl-I-methyl pyridinium ketoximes, Biochem. Pharmacol, 14,1471-1477 (1965); Kuca K, Picha J, Cabal J, Liska F, Synthesis of thethree monopyridinium oximes and evaluation of their potency toregenerate acetylcholinesterase inhibited by nerve agents, I App.Biomed, 2: 51-56 (2004); Van Hooidonk, C, Krauu G W, and Ginjaar, On thereactivity of organophosphorus compounds Part IV, The alkalinehydrolysis of some 0-phosphylated 2-pyridine oximes, Rec. Tray. Chim.,87, 673-686 (1968). Most enzymes lose activity with multiple turnovers.It is now possible to get multiple turnovers of AChE by incorporatingpolymeric oximes into the structure of the enzyme.

FIGS. 7A and 7B are a schematic diagrams contrasting the predictedreactivation pathways for enzyme polymer attached aldoximes andketoximes, respectively. The materials and methods for the chemicalreactions represented in FIGS. 7A and 7B are discussed in the recentwork by Vyas, S., Chem. Biol. Interact., (2008) (supra). The mechanismsshown in FIGS. 7A and 7B provide insight into the interactions ofaldoximes and ketoximes with OP compounds. As shown in FIG. 7B, theAChE-ketoxime polymer conjugate is schematically shown to remove thephosphoryl molecule from the AChE active site. The removal of theinhibitor thus regenerates the AChE and subsequently reacts theketoxime/phosphoryl polymer with water to self-regenerate the ketoximemolecule. The mechanism as shown in FIGS. 7A and B demonstrates that thestrong nucleophilic attack of the oxime functional group provided by theesterase polymer conjugate can remove the phosphoryl functional groupfrom the enzyme active site with relative ease resulting in theregenerated enzyme. The facility of the oxime functional group toregenerate the enzyme from the inhibited state is directly due to theproximal availability of the nucleophilic oxime to the active site. Thisis especially advantageous since the esterase delays clearance of theoxime antitoxin from the body and provides for the oxime antitoxinmolecule to remain, not only in the body but at the active site ready todetoxify and regenerate the enzyme upon inhibition. Similarly,regeneration of any enzyme active site inhibited by a phosphorylfunctional group would be a candidate for enzyme polyoxime conjugateregeneration. Furthermore, the delayed clearance of the oxime functionalgroup due to its covalent attachment to the enzyme provides prolongedavailability for the oxime antitoxin to detoxify not only OP toxinswithin the active site, but also those OP molecules free in the bodilyfluid.

The fact that there is an alkyl or aryl instead of a proton on thecarbon atom bound via a double bond to the —NOH moiety is a chemicalbasis for preventing the oxime from being converted to nitrile with itremaining an oxime (ketoxime in this case) after hydrolysis of thephosphoryl-ketoxime intermediate. The alkyl or aryl may prevent theBeckman rearrangement aided by the proton that converts the aldoximeinto a nitrile —CN group (as it happens with aldoximes that bear aproton on the respective carbon atom, see FIG. 7A). This reaction isharnessed with ketoxime-phosphoryl conjugates that are also formedduring reactivation. Importantly, ketoximes are usually less active thanaldoximes except for few cases such as the phenyl ketoxime analogue of2-PAM as reported Kuca K, Picha J, Cabal J, Liska F, Synthesis of thethree monopyridinium oximes and evaluation of their potency toregenerate acetylcholinesterase inhibited by nerve agents, J. App.Biomed., 2: 51-56 (2004). Ketoxime “Click” monomers can be synthesizedessentially as shown for the 2-PAM derivatives (see Tables 1 and 2).

In the first round, optimization of the self-reactivation ability of theesterase polymer conjugate is of concern with random co-polymers andblock copolymers of oximes and either spacer monomers (for example,DMAA) and/or environmentally responsive monomers as the variables. Invarious aspects, the number of monomers within a polyoxime may rangefrom about 1 to about 10 monomers, from about 1 to about 25 monomers,from about 10 to about 50 monomers, from about 1 to about 100 monomers,from about 25 to about 500 monomers, from about 500 to about 1 millionmonomers, or more than about 1 monomer, more than about 10 monomers,more than about 100 monomers, more than about 1000 monomers, or morethan about 1 million monomers. As used herein the term “environmentallyresponsive” refers to monomers that respond to environmental conditionssuch as pH, temperature, pressure, chemical concentrations, a change inlight energy, a change in electrical charge, or the like, andcombinations thereof. For example, a non-exhaustive list of suitableenvironmentally responsive monomers and their responding conditions isgiven in Table 3 shown below:

TABLE 3 Environmentally responsive monomers and their respondingconditions

Poly(N-isopropylacrylamide) Thermo-responsive polymer Lower criticalsolution temperature (LCST) ~33° C.

Poly(oligo(ethylene glycol) methyl ether methacrylate) Thermo-responsivepolymer Lower critical solution temperature (LCST): 35-82° C.

Poly(sulfobetaine methacrylate) Thermo-responsive polymer Upper criticalsolution temperature (UCST): 10-60° C.

Poly(N,N-dimethylaminoethyl methacrylate) Thermo- and pH responsivepolymer Lower critical solution temperature (LCST): 38° C. pH criticalpoint: ~9.0

Poly((methy)acrylate) pH responsive polymer pH critical point: ~2.0

Poly(N-acryloyl-6-aminohexanoic acid)) pH responsive polymer pH criticalpoint: ~4.5

It has been shown that polyDMAEMA conjugated to chymotrypsin predictablyaffects the local pH of the complex. Murata H, Cummings C S, Koepsel RR, Russell A J, Polymer-Based Protein Engineering Can Rationally TuneEnzyme Activity, pH-Dependence, and Stability, Biomacromolecules,10:14(6):1919-26 (2013).

Incorporating DMAEMA or other pH responsive monomers into the oximepolymer can raise (or lower) the local pH to match the pKa of the oximegroups (e.g., 7.8-8.1 for 2-PAM compounds) and increase their activityin physiological conditions. Thus, inclusion of pH responsive monomerswill be a point of emphasis for a second round of esterase polymerconjugate optimization. Additionally, the spacing between the oximefunctionality and the polymer backbone could affect access of the oximeto the active site and will be another optimization target with spacersbeing alkyl chains or short oligomers (e.g., PEG chains of 3-10monomers). Optimization utilizing spacers of alkyl chains or shortoligomers may include alkyl chains from about 1 carbon to about 100carbons, from about 2 carbons to about 75 carbons, from about 10 carbonsto about 50 carbons, from about 4 carbons to about 10 carbons, or forany range subsumed therein, for example, from about 3 carbons to about20 carbons. Further optimizations could include, enzyme conjugates withmultiple different polymers, polymers of multiple oximes, and variationsin the number of polymer per protein molecule. Other suitable polymersfor optimization of the esterase polymer conjugate may include non-oximemonomers as shown in Table 4 below:

TABLE 4 Non-oxime Monomers

N,N-dimethacrylamide

N-isopropylacrylamide thermo-responsive

Carboxyl acrylamide pH-responsive muco-adhesive

N,N-dimethyaminoethyl methacrylate thermo and pH-responsivemuco-adhesive

(meth)acrylate R: H or CH₃ pH-responsive muco-adhesive

2-hydroxylethyl methacrylate biocompatible

oligo(ethylene glycol) methyl ether methacrylate thermo-responsive

N-(2-Hydroxypropyl) methacrylamide biocompatible

Quaternary ammonium monomer muco-adhesive

Sulfobetain methacrylate thermo-responsive

For example, suitable non-oxime and oxime monomers may includealdoximes, ketoximes, muco-adhesion monomers, polyethylene glycol,bis-pyridinium oximes, N,N-dimethylacrylamide, N-isopropylacrylamide,(meth)acrylate, N,N-dimethylaminoethyl methacrylate, carboxylacrylamide, 2-hydroxylethylmethacrylate,N-(2-hydroxypropyl)methacrylamide, quaternary ammonium monomers,sulfobetain methacrylate, oligo(ethylene glycol) methyl ethermethacrylate, 2-PAM monomers, 4-PAM monomers, “Clickable” azidemonomers, and combinations thereof. It should be noted that most ofthese modifications could be performed by many suitable knownpolymerization methods, such as controlled radical polymerization or ona common base azide containing polymer using “Click” reactions with avariety of monomers. For example, “Click” reactions may be used with2-PAM monomers, 4-PAM monomers, “Clickable” azide monomers, andderivatives thereof as shown below in Table 5.

TABLE 5 Oxime and Clickable monomers

2-PAM monomer R₁: H or CH₃ R₂: O or NH

4-PAM monomer R₁: H or CH₃ R₂: O or NH

“Clickable” azide monomer R₁: H or CH₃ R₂: O or NH Click cycloadditionwith alkyne 2-PAM

Example 4

In a recent investigation of the modification of AChE with apoly[3-(N-2-methacryloyloxyethyl-N,N-dimethyl)ammonatopropanesulfonate](PMAPS) polymer, an enzyme activity assay determined a 1.6 fold enzymeactivity over the unmodified enzyme. It is believed that thedifferential in enzyme activity is due to the negative charges on thepolymer attracting the enzyme substrate through charge-chargeinteractions leading to a lower KM for the substrate (see FIG. 8). Thepoly(quaternary ammonium) (PQA) conjugate lost activity because thepositive charges repelled the substrate. Additionally, the quaternaryamine moieties on the polymer may block access to the active site thusacting as a competitive inhibitor.

AChE-pMAPS and AChE-pQA as shown in FIG. 8 were synthesized by“grafting-from” ATRP from an AChE-initiator conjugate using MAPS([3-(N-2-methacryloyloxyethyl-N,N-dimethyl)ammonatopropanesulfonate])and Quaternary ammonium (QA) monomer (2-(dimethylethylammonium)ethylmethacrylate), respectively. Details of the synthetic method aredisclosed in Example 2 herein. The enzyme activity of the AChE-pMAPS andAChE-pQA conjugates was determined using the following materials andmethods: Acetylthiocholine iodide (14.5 mM to 726 mM) and DTNB (497 mM)was added to sodium phosphate buffer (990 μL to 940 μL of 50 mM, pH7.4). Native AChE or conjugates solution (0.75 mM) was added to thesubstrate solution. The initial rate of hydrolysis of the acetylcholineiodide was monitored by recording the increase in absorption at 412 nmusing a UVNIS spectrometer. The Michaelis-Menten kinetic constants forthe reaction (kcat, KM, and kcat/KM) were determined by nonlinear curvefitting of plots of initial rate versus substrate concentration usingthe Enzfitter software.

Future Studies for Polymer-Enzyme Conjugate Molecular Analysis

Similar to the analysis of the esterase polymer conjugates describedherein, ongoing research not currently complete will include thefollowing studies. Engineered proteins will be analyzed for polymerconformation and size by gel permeation chromatography (GPC), dynamiclight scattering, and NMR. Conjugates will be tested in solution forenzymatic activity by measuring kinetic values and activity maxima fortemperature and pH. The susceptibility to inhibition by paraoxon and DFP(as surrogates for the OP toxins) as well as reactivation kinetics andenzyme stability will also be measured. Soluble forms of the conjugatedpolymers will be tested with native enzyme in parallel with thepolymer-protein conjugates to determine the influence of the polymerseparately from the conjugation. The influence of the various conjugatedpolymers will be determined by the performance of the polymer-AChEconjugate compared to native enzyme in solution.

The following enzyme assays will be performed as measures of therapeuticefficacy in vitro:

a) Evaluation of rate of direct interaction of oxime monomers, polymerbound oximes and enzyme-polymer-oxime conjugates with paraoxon anddiisopropyl fluorophosphate (DFP) sarin and VX in physiological buffersolution pH 7.4, 37° C. Initial activity of native AChE or 2-PAMconjugate may be monitored and then diluted to give a OD/sec of 0.2-0.4.The activity assay buffer may contain 50 mM sodium phosphate pH 7.4, 1mM acetylthiocholine iodide and 0.74 mM 5, 5′-Dithiobis (2-nitrobenzoicacid). The rate of hydrolysis of the substrate would be monitored forthe first 30 s by the increase in absorption at 412 nm in aspectrophotometer at 25° C.

b) In vitro reactivation studies with oxime monomers and polymer-boundoximes toward paraoxon and DFP, sarin and VX inhibition of AChE (AChEactivity will be measured by the Ellman method). The Ellman method usesEllman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) or DTNB) as achemical used to quantify the number or concentration of thiol groups ina sample. The method is named for George L. Ellman. Native AChE and theAChE-2-PAM conjugate (3.6×10⁻⁷ M) were inhibited with Paraoxon (1×10⁻⁶M). Aliquots will be sampled out into activity assay buffer containing50 mM sodium phosphate pH 7.4, 1 mM acetylthiocholine iodide and 0.74 mM5, 5′-Dithiobis (2-nitrobenzoic acid), and the optical density (OD)/secwill be monitored for a decrease in the OD/sec to achieve 90-95%inhibition. The inhibited enzyme would then be diluted 1:50 in either0.25 mM 2-PAM solution or into buffer. Aliquots will be sampled out intoactivity assay buffer and the Δ OD/sec will be monitored for an increasein the OD/min. The rate of hydrolysis of substrate would be monitored bythe increase in absorption, for example, at 412 nm.

c) In vitro inhibition and self-regeneration of AChE and BChE conjugatedto Oxime-Polymers in the presence of paraoxon and DFP, sarin and VX withvarious conjugate concentrations, and pH rate-profiles of reactivation.Experimental conditions and analysis will follow established protocolsfor determining the kinetics of simultaneous activity-inhibition assaysas reported in Estevez J, Vilanova E, Model equations for the kineticsof covalent irreversible enzyme inhibition and spontaneous reactivation:Esterases and organophosphorus compounds, Critical Reviews inToxicology, 39(5): 427-448 (2009).

To complete the inhibition of the enzyme, the native AChE and theAChE-2-PAM conjugate (3.6×10⁻⁷ M) will be inhibited with Paraoxon(1×10⁻⁶ M). Aliquots will be sampled out into activity assay buffermentioned above, and the OD/sec will be monitored for a decrease in theOD/sec to achieve 90-95% inhibition. To enable enzyme reactivation, thenative AChE and the 2-PAM conjugate (3.6×10⁻⁷ M) will be inhibited withParaoxon (1×10⁻⁶ M). Aliquots will be sampled out into activity assaybuffer mentioned above, monitoring the change in optical density persecond (OD/sec) for a decrease in OD/sec to achieve 90-95% inhibition.

Work has also begun using chymotrypsin (CT) in an enzyme polymerconjugate with poly(quaternary ammonium) (PQA). The PQA-CT conjugate wassynthesized by chemically attaching PQA chains to the surface of the CTenzyme. An enzyme inhibition assay was completed using the previouslydescribed materials and methods. The results of the preliminary studyare shown in FIG. 9. The concentration of the native enzyme was 3.9E−09Mand the PQA-CT conjugate was 1.89E−08M in the inhibition reaction. Theresults of the inhibition assay shows that the PQA-CT conjugate appearsto have self-regenerated in the presence of high concentrations ofdiisopropyl fluorophosphate (DFP).

The work to confirm and expand on the subject of utilizingchymotrypsin-polymer conjugates as regenerating bioscavengers is notcomplete at the time of the filing of this patent application.

The composition of the present invention may be used for treatment ofexposure to organophosphates and is suitable for human and mammalianveterinary use. The treatment may consist of a single dose or aplurality of doses over a period of time. The composition may beadministered by any suitable known method, including, withoutlimitation, by oral administration in liquid or tablet form, parenteraladministration (The term “parenteral” as used herein refers to modes ofadministration which include intraarticular injection, intravenousinjection, intrarterial injection, subcutaneous intramuscular injection,intrastemal injection, intraperitoneal injection, or infusion, includingdirect infusion to a target organ or organs), or by intranasal orinhalation techniques, depending on the amount and nature of theexposure and dosage deemed appropriate under the circumstances. Forexample, the appropriate dosage under circumstances of a single exposurearising, for example, from an accident where the amount oforganophosphate can be roughly determined may be calculated with greateraccuracy than the dosage needed under circumstances of mass exposurearising, for example, from chemical warfare or a terrorist attack wherethe amount of exposure may vary for each exposed individual. Massexposure and the need for rapid response times may require standarddosages that conform to an average body weight for exposed individuals.The dose of the composition will typically also vary depending on thesymptoms, age, gender, body weight, and extent of exposure, if known, ofthe individual patient (human or other mammal). Those skilled in the artcan set an appropriate dose and administration schedule if multipledoses are deemed necessary or desirable, taking into consideration theforegoing factors as well as the condition of the patient, the number ofpatients, and the particular route of administration. The compositionsof the present invention designed for pharmaceutical uses will beformulated and dosed in a fashion consistent with good medical practice,taking into account the clinical condition of the individual patient,the site of delivery, the method of administration, the scheduling ofadministration, and other factors known to practitioners. As statedpreviously, the required dose of the regenerating esterase polyoximeconjugate according to any of the aspects described herein, is expectedto be significantly reduced by, for example, 1-2 orders of magnitude, ascompared to stoichiometric free oximes and/or free cholinesterasesheretofore reported. The “effective amount” for purposes herein is thusdetermined by such considerations. In various aspects, the effectiveamount of the composition administered to a patient suffering fromorganophosphate toxin exposure is an amount necessary to reduce andpreferably eliminate, the inhibition of the normal esterase function.Reduction short of elimination is preferably sufficient to prevent deathof the individual due to the esterase inhibition. The dosage amountrelative to the body weight of the individual patient and route ofadministration will be subject to therapeutic discretion.

In various aspects, the composition may be used with a pharmaceuticallyacceptable salt. As used herein, “pharmaceutically acceptable” meanscompositions and molecular forms and ingredients of the compositionsthat are physiologically tolerable and do not produce toxic reactionswhen administered to a mammal. Pharmaceutically acceptable compositionsmay be listed in the U.S. or other recognized pharmacopeia for use withmammals and in particular, for use with humans.

Some pharmaceutically acceptable salts are acetate, adipate, aspartate,benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate,citrate, formate, fumarate, gluconate, glucuronate, hexafluorophosphate,hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide,lactate, malate, maleate, malonate, mandelates, mesylate,methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, oxalate,palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate,pyroglutamate, salicylate, saccharate, stearate, succinate, sulfonate,stannate, tartrate, tosylate, and trifluoroacetate salts. Known basicsalts may also be used in certain aspects.

In various aspects, the compositions of the present invention may beadministered alone or in combination with a pharmaceutically acceptablecarrier or diluent by any of the routes of administration describedherein. In various aspects, the compositions of the present inventionmay be administered alone or in combination with a pharmaceuticallyacceptable additive or excipient. For example, any one or more of theabove mentioned salts may be added to the carrier or diluent, or incertain aspects, may be incorporated in one or more of the monomers ofthe polymers bound to the esterase. The compositions may be administeredin single or multiple doses once or over a period of time. In variousaspects, the compositions may be administered as part of a combinationtherapy with another pharmaceutical agent.

Such carriers may include solid diluents or fillers, sterile aqueousmedia and various non-toxic organic solvents, etc. Moreover, oralpharmaceutical compositions can be suitably sweetened and/or flavored.In general, the therapeutically effective compounds of this inventionare present in such dosage forms at concentration levels ranging about1.0% to about 98% by weight, or from about 2% to about 95% by weight, orabout 5.0% or 10% to about 90%, 85%, 80%, 75%, 70%, 65%, 60%, or 50% byweight.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch, together with granulation binders likepolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are preferred for tableting purposes. Solid compositions of asimilar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions are desired for oral administration, the activecompositions may be combined with various sweetening or flavoringagents, coloring matter or dyes, and, if so desired, emulsifying and/orsuspending agents known to those skilled in the pharmaceutical fields,as well, together with such diluents as water, ethanol, propyleneglycol, glycerin and various like combinations thereof.

For parenteral administration, a compound according to any aspect of thepresent invention may be suspended in solutions or suspensions of apharmaceutically acceptable oil or aqueous propylene glycol. The aqueoussolutions should preferably be compatible with the physiological pH ofthe individual recipient, for example, greater than a neutral, andpreferably greater than pH 8, but less than a deleterious level. Theliquid diluents are preferably isotonic. All solutions must be preparedunder sterile conditions by standard pharmaceutical techniqueswell-known to those skilled in the art. Oily solutions are preferred forintra-articular, intra-muscular and subcutaneous injection. Aqueoussolutions are preferred for intravenous injection. Typically, thecarriers will be water or saline which will be sterile and pyrogen free.The compositions of the present invention, in various aspects, arebelieved to be well suited to formulation in aqueous carriers such assterile pyrogen free water, saline or other isotonic solutions. Some orall of the compositions described herein for pharmaceutical applicationsmay be formulated well in advance in aqueous form, for instance, weeksor months or longer time periods before being dispensed.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulationappropriate for the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules, vials or syringes, and may bestored in a freeze-dried (lyophilized) condition requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders.

It should be understood that this disclosure is not limited to thevarious aspects or embodiments disclosed herein, and it is intended tocover modifications that are within the spirit and scope of theinvention, as defined by the claims.

What is claimed is:
 1. A method comprising administering a bioscavengerto an individual identified as having been exposed to an organophosphatetoxin, wherein the bioscavenger comprises at least one polymercovalently conjugated to an esterase, and wherein the at least onepolymer comprises a plurality of oxime functional groups.
 2. The methodof claim 1, wherein, upon exposure of the bioscavenger to anorganophosphate toxin, the method further comprises reacting at leastone of the plurality of oxime functional groups with at least onecovalently inhibited residue of the esterase to detoxify theorganophosphate toxin and regenerate the bioscavenger.
 3. The method ofclaim 1, wherein the plurality of oxime functional groups comprises anoxime functional group positioned to exert, in use, a nucleophilicattack on a phosphoryl functional group of an inhibitor when thephosphoryl functional group is attached to an active site of theesterase, and wherein said nucleophilic attack results in removal of thephosphoryl functional group from the active site.
 4. The method of claim1, wherein the esterase comprises chymotrypsin and the plurality ofoxime functional groups comprises an aldoxime.